The Molecular Probes® Handbook A GUIDE TO ... 13 Probes for Lipids and Membranes Molecular Probes Handbook A Guide to Fluorescent Probes and Labeling Technologies 11th Edition (2010)

CHAPTER 13

Probes for Lipids and Membranes

Molecular Probes HandbookA Guide to Fluorescent Probes and Labeling Technologies

11th Edition (2010)

CHAPTER 1

Fluorophores and Their Amine-Reactive Derivatives

The Molecular Probes HandbookA GUIDE TO FLUORESCENT PROBES AND LABELING TECHNOLOGIES11th Edition (2010)

Molecular Probes Resources

Molecular Probes Handbook (online version)Comprehensive guide to uorescent probes and labeling technologies

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Fluorescence SpectraViewerIdentify compatible sets of uorescent dyes and cell structure probes

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BioProbes Journal of Cell Biology ApplicationsAward-winning magazine highlighting cell biology products and applications

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Access all Molecular Probes educational resources at lifetechnologies.com/mpeducate

Molecular Probes ResourcesMolecular Probes Handbook (online version)Comprehensive guide to fl uorescent probes and labeling technologiesthermofi sher.com/handbook

Molecular Probes Fluorescence SpectraViewerIdentify compatible sets of fl uorescent dyes and cell structure probesthermofi sher.com/spectraviewer

BioProbes Journal of Cell Biology ApplicationsAward-winning magazine highlighting cell biology products and applicationsthermofi sher.com/bioprobes

Access all Molecular Probes educational resources at thermofi sher.com/probes

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The Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling TechnologiesIMPORTANT NOTICE: The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

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CHAPTER 13

Probes for Lipids and Membranes13.1 Introduction to Membrane Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 547Fluorescent and Biotinylated Membrane Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 547

Fluorescent Analogs of Natural Lipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 547

Other Lipophilic and Amphiphilic Fluorescent Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 548

Other Probes for Studying Cell Membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 548

13.2 Fatty Acid Analogs and Phospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 549Fluorescent Fatty Acid Analogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 549

BODIPY Fatty Acids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 549

NBD Fatty Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 551

Pyrene Fatty Acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 551

Dansyl Undecanoic Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 551

cis-Parinaric Acid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 551

ADIFAB Fatty Acid Indicator . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 553Phospholipids with BODIPY DyeLabeled Acyl Chains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 554

BODIPY Glycerophospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 554

Applications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 556

Phospholipid with DPH-Labeled Acyl Chain. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 557Properties. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 557

Applications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 557

Phospholipids with NBD-Labeled Acyl Chains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 557Properties. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 557

Applications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 557

Phospholipids with Pyrene-Labeled Acyl Chains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 558Properties. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 558

Applications. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 558

Phospholipids with a Fluorescent or Biotinylated Head Group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 559Phospholipid with a Dansyl-Labeled Head Group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 559

Phospholipid with a Marina Blue DyeLabeled Head Group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 559

Phospholipid with a Pacic Blue DyeLabeled Head Group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 559

Phospholipid with an NBD-Labeled Head Group. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 559

Phospholipid with a Fluorescein-Labeled Head Group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 559

Phospholipid with an Oregon Green 488 DyeLabeled Head Group. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 560

Phospholipid with a BODIPY FL DyeLabeled Head Group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 560

Phospholipids with a Rhodamine or Texas Red DyeLabeled Head Group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 560

Phospholipids with a Biotinylated Head Group . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 560

LipidTOX Phospholipid and Neutral Lipid Stains for High-Content Screening . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 561HCS LipidTOX Phospholipidosis Detection Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 561

HCS LipidTOX Neutral Lipid Stains. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 561

HCS LipidTOX Phospholipidosis and Steatosis Detection Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 562

Data Table 13.2 Fatty Acid Analogs and Phospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 563Product List 13.2 Fatty Acid Analogs and Phospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 565

13.3 Sphingolipids, Steroids, Lipopolysaccharides and Related Probes . . . . . . . . . . . . . . . . . . . . 566Sphingolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 566

Structure and Activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 566

BODIPY Sphingolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 567

The Molecular Probes Handbook: A Guide to Fluorescent Probes and Labeling Technologies

IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.

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Chapter 13 Probes for Lipids and Membranes

NBD Sphingolipids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 568

Vybrant Lipid Raft Labeling Kits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 568

Amplex Red Sphingomyelinase Assay Kit. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 569

Steroids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 569BODIPY Cholesteryl Esters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 569

Side ChainModied Cholesterol Analog . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 570

Amplex Red Cholesterol Assay Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 570

Fluorescent Triacylglycerol. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571Lipopolysaccharides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571

Fluorescent Lipopolysaccharides. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 571

Pro-Q Emerald 300 Lipopolysaccharide Gel Stain Kit . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 572

Data Table 13.3 Sphingolipids, Steroids, Lipopolysaccharides and Related Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 573Product List 13.3 Sphingolipids, Steroids, Lipopolysaccharides and Related Probes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 574

13.4 Dialkylcarbocyanine and Dialkylaminostyryl Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 575Dialkylcarbocyanine Probes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 575

DiI, DiO, DiD, DiR and Analogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 575

Spectral Properties of Dialkylcarbocyanines . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 575

Substituted DiI and DiO Derivatives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 576

DiI and DiO as Probes of Membrane Structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 577

DiI and DiO as Probes of Membrane Dynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 577

Dialkylaminostyryl Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 577Data Table 13.4 Dialkylcarbocyanine and Dialkylaminostyryl Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 578Product List 13.4 Dialkylcarbocyanine and Dialkylaminostyryl Probes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579

13.5 Other Nonpolar and Amphiphilic Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579Amphiphilic Rhodamine, Fluorescein and Coumarin Derivatives. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579

Octadecyl Rhodamine B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579

Amphiphilic Fluoresceins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 580

Amphiphilic Coumarin. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 580

DPH and DPH Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 580Diphenylhexatriene (DPH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 580

TMA-DPH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581

Nonpolar BODIPY Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581BODIPY Fluorophores. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 581

BODIPY FL C5-Ceramide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 582

CellTrace BODIPY TR Methyl Ester . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 582

Pyrene, Nile Red and Bimane Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 582Nonpolar Pyrene Probe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 582

Nile Red . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 582

Bimane Azide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 583

LipidTOX Neutral Lipid Stains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 583Membrane Probes with Environment-Sensitive Spectral Shifts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584

Prodan and Laurdan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584

Dapoxyl Derivative . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584

Anilinonaphthalenesulfonate (ANS) and Related Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 584

Bis-ANS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585

DCVJ. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 585

Data Table 13.5 Other Nonpolar and Amphiphilic Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 586Product List 13.5 Other Nonpolar and Amphiphilic Probes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 587


IMPORTANT NOTICE : The products described in this manual are covered by one or more Limited Use Label License(s). Please refer to the Appendix on page 971 and Master Product List on page 975. Products are For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.thermofisher.com/probes




Section 13.1 Introduction to Membrane Probes

13.1 Introduction to Membrane Probes

Fluorescent and Biotinylated Membrane Probese plasma membranes and intracellular membranes of live cells and the articial mem-

branes of liposomes represent a signicant area of application for uorescent probes. Membrane probes include uorescent analogs of natural lipids, as well as lipophilic organic dyes that have little structural resemblance to natural biomolecules. We oer a wide range of both types of membrane probes. ese probes are used for structural and biophysical analysis of membranes, for following lipid transport and metabolism in live cells (Figure 13.1.1) and for investigating synaptosome recycling (Section 16.1) and lipid-mediated signal transduction processes (Chapter 17). Due to their low toxicity and stable retention, some lipid probes are particularly useful for long-term cell tracing (Section 14.4). Other, slightly less lipophilic probes are used as membrane markers of endocytosis and exocytosis (Section 16.1).

Fluorescent Analogs of Natural LipidsWe oer uorescent and, in a few cases, biotinylated analogs of ve naturally occurring lipid

classesphospholipids, sphingolipids (including ceramides), fatty acids, triglycerides and ste-roids. Phospholipids are the principal building blocks of cell membranes. Most phospholipids are derivatives of glycerol comprising two fatty acyl residues (nonpolar tails) and a single phosphate ester substituent (polar head group). Despite their overall structural similarity (Figure 13.1.2), natural phospholipids exhibit subtle dierences in their fatty acid compositions, degree of acyl

Figure 13.1.1 The cytoplasm of a live zebrash embryo labeled with the green-uorescent lipophilic tracer BODIPY 505/515 (D3921). The image was contributed by Arantza Barrios, University College, London.

Figure 13.1.2 A) Phosphatidylcholines, phosphatidylinositols and phosphatidic acids are examples of glycerolipids derived from glycerol. B) Sphingomyelins, ceramides and cerebrosides are examples of sphingolipids derived from sphingosine. In all the structures shown, R represents the hydrocarbon tail portion of a fatty acid residue.

Glycerol

Sphingosine

Sphingomyelin Cerebroside

Phosphatidylcholine Phosphatidic Acid

Ceramide

Phosphatidylinositol

P

O

O-

(CH3)3NCH2CH2O

C

R

O

+

OH

OH

HO

O

HO

O

R

C

C

R

O

O CH2O

CH CH2O

O CH2O

CH CH2O

O CH2O

CH CH2O

HOCH2 CH CH

NH2 OH

CH CH(CH2)12CH3

CH2 CH CH

NH OH

CH CH(CH2)12CH3O O CH(CH2)12CH3CH

OHNH

CHCHCH2

HOCH2 CH CH

NH OH

CH CH(CH2)12CH3

CH

OH

HOCH2 CH2OH

+P

O

O-

(CH3)3NCH2CH2O

O

R

C C

R

O

P

O

O-

-O

O

R

C C

R

O

P

O

O-

O

R

C

O

OH

OH

HO

OH OH

C

R

O

A

B



thermofisher.com/probes




Section 13.1 Introduction to Membrane Probes

REFERENCES1. Nat Rev Mol Cell Biol (2008) 9:112; 2. Biochim Biophys Acta (1994) 1212:26; 3. J Lipid Res (1998) 39:467; 4. Chem Phys Lipids (1991) 58:111; 5. Annu Rev Biochem (1998) 67:27; 6. Biochim Biophys Acta (1991) 1082:113; 7. Ann N Y Acad Sci (1998) 845:57; 8. Nat Rev Mol Cell Biol (2008) 9:125.

chain unsaturation and type of polar head group.1 ese dierences can produce signicant variations in membrane physical properties, in the location of phospholipids in a lipid bilayer and in their biological activ-ity. Fluorescent phospholipid analogs (Section 13.2) can be classied according to where the uorophore is attached. e uorophore can be attached to one (or both) of the fatty acyl chains or to the polar head group. e attachment position of the uorophore determines whether it is located in the nonpolar interior or at the water/lipid interface when the phospholipid analog is incorporated into a lipid bilayer membrane.

Fatty acids are the building blocks for a diverse set of biomol-ecules. Some fatty acids (e.g., arachidonic acid) are important in cell signaling.2 Fatty acids are liberated by the enzymatic action of phospho-lipase A on phospholipids (Section 17.4) and also by various other lipas-es. Fluorescent fatty acids can oen be used interchangeably with the corresponding phospholipids as membrane probes; however, fatty acids transfer more readily between aqueous and lipid phases.3 Although fat-ty acids are ionized at neutral pH in water (pKa ~5), their pKa is typically ~7 in membranes, and thus a signicant fraction of membrane-bound fatty acids are neutral species.3 Certain uorescent fatty acids (Section 13.2) are readily metabolized by live cells to phospholipids, mono-, di- and triacylglycerols, cholesteryl esters and other lipid derivatives.4

Sphingolipids play critical roles in processes such as proliferation, apoptosis, signal transduction and molecular recognition at cell sur-faces.1,5,6 Defects in the lysosomal breakdown of sphingolipids are the underlying cause of lipid storage disorders such as NiemannPick, TaySachs, Krabbe and Gaucher diseases. e sphingolipids described in Section 13.3 include ceramides, sphingomyelins, glycosylceramides (ce-rebrosides) and gangliosides. e structural backbone of sphingolipids is the lipophilic aminodialcohol sphingosine (2-amino-4-octadecen-1,3-diol, Figure 13.1.2) to which a single fatty acid residue is attached via an amide linkage. Our uorescent analogs of sphingolipids are prepared by replacing the natural amide-linked fatty acid with a uorescent ana-log. Sphingolipids with an unmodied hydroxyl group in the 1-position are classied as ceramides. As part of the lipid-sorting process in cells, ceramides are glycosylated to cerebrosides (Figure 13.1.2) or converted to sphingomyelins (Figure 13.1.2) in the Golgi complex. Glycosylated

Figure 13.1.3 The neuronal tracer DiI (D282, D3911) used as a diagnostic tool to evaluate patterns of innervation in newborn mouse cochlea. The larger image is of a mutant cochlea and the inset is of a wild-type cochlea. Image contributed by Bernd Fritzsch, Creighton University, and L. Reichardt and I. Farinas, Howard Hughes Medical Institute, San Francisco.

sphingolipids (cerebrosides and gangliosides) occur in the plasma mem-branes of all eukaryotic cells and are involved in cell recognition, signal transduction and modulation of receptor function.7 Gangliosides have complex oligosaccharide head groups containing at least one sialic acid residue in place of the single galactose or glucose residues of cerebrosides.

Fluorescent cholesteryl esters and triglycerides (Section 13.3) can be used as structural probes and transport markers for these important lipid constituents of membranes and lipoproteins.8 ey may also serve as uorescent substrates for lipases and lipid-transfer proteins and can be incorporated into low-density lipoproteins (LDL, Section 16.1).

Other Lipophilic and Amphiphilic Fluorescent Probese probes described in Section 13.4 and Section 13.5 are not ana-

logs of any particular biological lipid class, but they have a general struc-tural resemblance that facilitates labeling of membranes, lipoproteins and other lipid-based molecular assemblies. Particularly notable mem-bers of this group are the lipophilic carbocyanines DiI (Figure 13.1.3), DiO, DiD and DiR, the lipid uidity probes DPH and TMA-DPH and the membrane-surface probes ANS and laurdan. ese probes generally have limited water solubility and exhibit substantially enhanced uo-rescence upon partition into lipid environments. ey can be classied as either amphiphilic (having both polar and nonpolar structural ele-ments) or neutral (lacking charges and most soluble in very nonpolar environments). We use similar neutral lipophilic dyes for internal stain-ing of our uorescent polystyrene microspheres (Section 6.5).

Other Probes for Studying Cell MembranesIn addition to the lipophilic probes described in this chapter, we

have available the following products for studying the properties and functions of cell membranes: Moderately lipophilic stains for the endoplasmic reticulum and

Golgi apparatus (Section 12.4) FM dyesamphiphilic probes for cell membrane labeling (Section

14.4, Section 16.1) CellLight Plasma Membrane-CFP, CellLight Plasma Membrane-

GFP and CellLight Plasma Membrane-RFP, which are BacMam 2.0 vectors encoding uorescent proteins targeted to the plasma membrane (C10606, C10607, C10608; Section 14.4)

Alexa Fluor dyelabeled cholera toxin subunit B conjugates for labeling lipid ras (Section 14.7)

Annexin V conjugates for detection of phosphatidylserine expo-sure in apoptotic cell membranes (Section 15.5)

Fluorescent and uorogenic phospholipase A substrates (Section 17.4) Amplex Red Phosphatidylcholine-Specic Phospholipase C Assay

Kit and Amplex Red Phospholipase D Assay Kit (A12218, A12219; Section 17.4)

Antibodies to phosphatidylinositol phosphates (Section 17.4) Lipophilic pH indicators (Section 20.4) Membrane potentialsensitive probes (Section 22.2, Section 22.3)






Section 13.2 Fatty Acid Analogs and Phospholipids

13.2 Fatty Acid Analogs and Phospholipidse probes in this section and in Section 13.3 bear some structural

resemblance to natural lipids. Included in this section are uorescent fatty acid analogs, as well as phospholipids wherein one or both fatty acid esters are replaced by uorescent fatty acid esters. e uorophores in these probes tend to remain buried in the hydrophobic interior of lipid bilayer membranes.1,2 In this location, they are sensitive to mem-brane properties such as lipid uidity, lateral domain formation and structural perturbation by proteins, drugs and other additives. Also included in this section are several head groupmodied phospholipid analogs incorporating a uorophore or biotin (Table 13.1).

Sphingolipids, steroids and lipopolysaccharides are discussed in Section 13.3. Important applications of the uorescent phosphati-dylinositol derivatives as probes for signal transduction and various uorescent phospholipids as phospholipase substrates are further de-scribed in Section 17.4. A review of uorescent lipid probes and their use in biological and biophysical research has been published.3

Fluorescent Fatty Acid AnalogsOur uorescent fatty acid analogs have a uorophore linked within

the fatty acid chain or, more commonly, at the terminal (omega) carbon atom that is furthest from the carboxylate moiety. Although uorescent fatty acid analogs are sometimes used as direct probes for membranes

Table 13.1 Phospholipids with labeled head groups.

Label (Ex/Em)* Cat. No.Dansyl (336/517) D57Marina Blue (365/460) M12652Pacic Blue (410/455) P22652NBD (463/536) N360Fluorescein (496/519) F362Oregon Green 488 (501/526) O12650BODIPY FL (505/511) D3800BODIPY 530/550 D3815Tetramethylrhodamine (540/566) T1391Lissamine rhodamine (560/581) L1392Texas Red (582/601) T1395MPBiotin (





fatty acids also exhibit excimer formation but their emission is at much shorter wavelengths than that of the BODIPY dyes and they are there-fore less suitable for the study of live cells.

e uorophores in our current selection of BODIPY fatty acids and their approximate absorption/emission maxima (in nm) are:

BODIPY 503/512 (BODIPY FL; D3821, Figure 13.2.3; D3822; D3834; D3862)

BODIPY 500/510 (D3823, B3824, D3825; Figure 13.2.4) BODIPY 530/550 (D3832, Figure 13.2.5) BODIPY 558/568 (D3835, Figure 13.2.6) BODIPY 581/591 (D3861)

BODIPY fatty acids are synthetic precursors to a wide variety of uorescent phospholipids (described below), as well as several impor-tant sphingolipid probes described in Section 13.3. Some BODIPY fat-ty acids are readily metabolized by live cells to phospholipids, di- and triacylglycerols, cholesteryl esters and other natural lipids.69 Analysis of cellular lipid extracts by HPLC has shown that glycerophosphocho-lines constitute more than 90% of the products of biosynthetic incor-poration of BODIPY 500/510 dodecanoic acid (D3823) by BHK cells.5

e three BODIPY 500/510 probes form a unique series in which the green-uorescent uorophore is located within the fatty acid chain at dierent distances from the terminal carboxylate group.4 e overall length of the probe is constant and, including the uorophore, is about equivalent to that of an 18-carbon fatty acid (Figure 13.2.4).

BODIPY 581/591 undecanoic acid (D3861) is particularly use-ful for detecting reactive oxygen species in cells and membranes.1013 Oxidation of the polyunsaturated butadienyl portion of the BODIPY 581/591 dye (Figure 13.2.7) truncates the conjugated -electron system, resulting in a shi of the uorescence emission peak from ~590 nm to ~510 nm.10,13,14 is oxidation response mechanism is similar to that of the naturally occurring polyunsaturated fatty acid cis-parinaric acid. In comparison to cis-parinaric acid, advantages of BODIPY 581/591 un-decanoic acid include:

Long-wavelength excitation, compatible with confocal laser-scan-ning microscopes and ow cytometers

Avoidance of photooxidation eects induced by ultraviolet excitation

Less interference by colored oxidant and antioxidant additives when detecting probe uorescence 12

Greater resistance to spontaneous oxidation Red-to-green uorescence shi, allowing the use of uorescence

ratio detection methods 10,13

Figure 13.2.3 4,4-Diuoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-hexadecanoic acid (BODIPY FL C16, D3821).

NB

NFF (CH2)15H3C

H3C

C OHO

Figure 13.2.4 Structural representations showing the positional shift of the uorophore with respect to the terminal carboxyl group in a homologous series of BODIPY 500/510 fatty acids (D3823, B3824, D3825).

C1-BODIPY 500/510 C12

NB

N

FFC

O

OH

NB

N

FF C OH

O

NB

N

FF C OH

O

C4-BODIPY 500/510 C9

C8-BODIPY 500/510 C5

Figure 13.2.5 4,4-Diuoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (BODIPY 530/550 C12, D3832).

NB

NFF (CH2)11 C OH

O

Figure 13.2.6 4,4-Diuoro-5-(2-thienyl)-4-bora-3a,4a-diaza-s-indacene-3-dodecanoic acid (BODIPY 558/568 C12, D3835).

NB

NFF (CH2)11 C OH

O

Figure 13.2.7 4,4-Diuoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (BODIPY 581/591 C11, D3861).







An alternative technique for detecting lipid peroxidation utilizes the oxidation-induced de-crease of concentration-dependent excimer formation by BODIPY FL dyelabeled fatty acids.15

NBD Fatty AcidsFluorescence of the nitrobenzoxadiazole (NBD) uorophore is highly sensitive to its envi-

ronment. Although it is moderately uorescent in aprotic solvents, in aqueous solvents it is al-most nonuorescent.16 e NBD uorophore is moderately polar and both its homologous 6-car-bon and 12-carbon fatty acid analogs (N316, Figure 13.2.8; N678) and the phospholipids derived from these probes (N3786, N3787) tend to sense the lipidwater interface region of membranes instead of the hydrophobic interior 17 (see part B of Figure 13.2.1). e environmental sensitivity of NBD fatty acids can be usefully exploited to probe the ligand-binding sites of fatty acid and sterol carrier proteins.18 NBD fatty acids are not well metabolized by live cells.9,19

Pyrene Fatty Acidse hydrophobic pyrene uorophore is readily accommodated within the membrane.20

-Pyrene derivatives of longer-chain fatty acids (Figure 13.2.9) were rst described by Galla and Sackmann in 1975.21 We oer pyrene derivatives of the 4-, 10-, 12- and 16-carbon fatty acids (P1903MP, P31, P96, P243, respectively). Pyrenebutanoic acidfrequently called pyrenebutyric acid (P1903MP)has rarely been used as a membrane probe; however, its conjugates have ex-ceptionally long excited-state lifetimes ( >100 nanoseconds) and are consequently useful for time-resolved uorescence immunoassays and nucleic acid detection.22,23 e long excited-state lifetime of pyrenebutyric acid also makes it useful as a probe for oxygen in cells 2427 and lipid vesicles.28

Pyrene derivatives form excited-state dimers (excimers) with red-shied uorescence emis-sion 2931 (Figure 13.2.10). Pyrene excimers can even form when two pyrenes are tethered by a short trimethine spacer, as in 1,3-bis-(1-pyrenyl)propane (B311, Section 13.5). Pyrene excimer formation is commonly exploited for assaying membrane fusion 32,33 (Lipid-Mixing Assays of Membrane FusionNote 13.1) and for detecting lipid domain formation.3436 Pyrene fatty ac-ids are metabolically incorporated into phospholipids, di- and tri-acylglycerols and cholesteryl esters by live cells.19,37,38

Other uses of pyrene fatty acids include:

Detecting lipidprotein interactions 9,40

Inducing photodynamic damage 41,42

Investigating phospholipase A2 action on lipid assemblies 4345

Studying lipid transport mechanisms and transfer proteins 4648

Synthesizing uorescent sphingolipid probes 4952

Dansyl Undecanoic AcidDansyl undecanoic acid (DAUDA, D94; Figure 13.2.11) incorporates a polar, environment-

sensitive dansyl uorophore that preferentially locates in the polar headgroup region of lipid bilayer membranes.53 DAUDA exhibits a 60-fold uorescence enhancement and a large emission spectral shi to shorter wavelengths on binding to certain proteins.54 is property has been exploited to analyze fatty acidbinding proteins 5457 and also to develop a uorometric phospho-lipase A2 assay (Section 17.4) based on competitive fatty acid displacement.5861

cis-Parinaric Acide naturally occurring polyunsaturated fatty acid cis-parinaric acid (P36005, Figure

13.2.12) was initially developed as a membrane probe by Hudson and co-workers and published in 1975.62 cis-Parinaric acid is the closest structural analog of intrinsic membrane lipids among currently available uorescent probes (Figure 13.2.1). e chemical and physical properties of cis-parinaric acid have been well characterized. e lowest absorption band of cis-parinaric acid has two main peaks around 300 nm and 320 nm, with a high extinction coecient. cis-Parinaric acid oers several experimentally advantageous optical properties, including a very large uores-cence Stokes shi (~100 nm) and an almost complete lack of uorescence in water. In addition, the uorescence decay lifetime of cis-parinaric acid varies from 1 to ~40 nanoseconds, depend-ing on the molecular packing density in phospholipid bilayers. Consequently, minutely detailed information on lipid-bilayer dynamics can be obtained.

Figure 13.2.8 NBD-X (6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoic acid; N316).

NO

N

NO2

NH(CH2)5 C OHO

Figure 13.2.9 1-Pyrenedodecanoic acid (P96).

Figure 13.2.10 Excimer formation by pyrene in ethanol. Spectra are normalized to the 371.5 nm peak of the monomer. All spectra are essentially identical below 400 nm after normalization. Spectra are as follows: 1) 2 mM pyrene, purged with argon to remove oxygen; 2) 2 mM pyrene, air-equilibrated; 3) 0.5 mM pyrene (argon-purged); and 4) 2 M pyrene (argon-purged). The monomer-to-excimer ratio (371.5 nm/470 nm) is dependent on both pyrene concentra-tion and the excited-state lifetime, which is variable because of quenching by oxygen.

Fluo

resc

ence

em

issi

onWavelength (nm)

350 450400 500 550 600

1

2

3

4

Figure 13.2.11 11-((5-Dimethylaminonaphthalene-1-sul-fonyl)amino)undecanoic acid (DAUDA, D94).

Figure 13.2.12 cis-Parinaric acid (P36005).

(CH2) CO

OHCC

CCCC

CCH

CH3CH2

H H

HH

HH

H








Figure 1 Pictorial representation of a lipid-mixing assay based on uores-cence resonance energy transfer (FRET). The average spatial separation of the donor (D) and acceptor (A) lipid probes increases upon fusion of labeled membranes with unlabeled membranes, resulting in decreased eciency of proximity-dependent FRET (represented by yellow arrows). Decreased FRET eciency is registered by increased donor uorescence intensity and decreased acceptor uorescence intensity.

Figure 2 Pictorial representation of a lipid-mixing assay based on uo-rescence self-quenching. Fluorescence of octadecyl rhodamine B (O246), incorporated at >1:100 with respect to host membrane lipids, is quenched due to dyedye interactions. Fusion with unlabeled membranes causes dispersion of the probe, resulting in a uorescence increase that is repre-sented here by a color change from black to green.

Figure 4 Pictorial representation of a lipid-mixing assay based on pyrene excimer formation. Locally concentrated pyrene-labeled lipid probes emit red-shifted uorescence due to formation of excimers (excited-state dimers). Probe dilution by unlabeled lipids as a result of membrane fusion is registered by the replacement of excimer emission by blue-shifted monomer uorescence.

Pyrene excimer uorescence~470 nm

Fusion

OO O O OO O O

+

Pyrene monomer uorescence~400 nm

O OO O O O O O

Fluorometric methods for assaying membrane fusion exploit processes, such as nonradiative energy transfer, uorescence quenching and pyrene excimer formation, that are dependent on probe concentration.18 Assays of membrane fusion report either the mixing of membrane lipids (described here) or the mixing of the aqueous contents of the fused entities (Assays of Volume Change, Membrane Fusion and Membrane PermeabilityNote 14.3). Chapter 13 describes additional methods for detecting membrane fusion based on image analysis.

NBDRhodamine Energy TransferPrinciple: Struck, Hoekstra and Pagano introduced lipid-mixing assays based

on NBDrhodamine energy transfer.9 In this method (Figure 1), membranes labeled with a combination of uorescence energy transfer donor and accep-tor lipid probestypically NBD-PE and N-Rh-PE (N360, L1392; Section 13.2), respectivelyare mixed with unlabeled membranes. Fluorescence resonance energy transfer (FRET), detected as rhodamine emission at ~585 nm resulting from NBD excitation at ~470 nm, decreases when the average spatial separation of the probes is increased upon fusion of labeled membranes with unlabeled membranes. The reverse detection scheme, in which FRET increases upon fu-sion of membranes that have been separately labeled with donor and acceptor probes, has also proven to be a useful lipid-mixing assay.10

Applications: Applications of the NBDrhodamine assay are described in footnoted references.1120

Octadecyl Rhodamine B Self-QuenchingPrinciple: Lipid-mixing assays based on self-quenching of octadecyl

rhodamine B (R18, O246; Section 13.5) were originally described by Hoekstra and co-workers.21 Octadecyl rhodamine B self-quenching occurs when the probe is incorporated into membrane lipids at concentrations of 110 mole percent.22 Unlike phospholipid analogs, octadecyl rhodamine B can readily be introduced into existing membranes in large amounts. Fusion with unlabeled membranes results in dilution of the probe, which is accompanied by increasing uorescence 23,24 (excitation/emission maxima 560/590 nm) (Figure 2). The assay may be compromised by eects such as spontaneous transfer of the probe to unlabeled membranes, quenching of uorescence by proteins and probe-related inactivation of viruses; the prevalence of these eects is currently debated.2527

Applications: The octadecyl rhodamine B self-quenching assay is exten-sively used for detecting viruscell fusion.2839

Pyrene Excimer FormationPrinciple: Pyrene-labeled fatty acids (e.g., P31, P96, P243; Section 13.2) can

be biosynthetically incorporated into viruses and cells in sucient quantities to produce the degree of labeling required for long-wavelength pyrene excimer uorescence (Figure 3). This excimer uorescence is diminished upon fusion of labeled membranes with unlabeled membranes (Figure 4). Fusion can be monitored by following the increase in the ratio of monomer (~400 nm) to excimer (~470 nm) emission, with excitation at about 340 nm. This method ap-pears to circumvent some of the potential artifacts of the octadecyl rhodamine B self-quenching technique 26 and, therefore, provides a useful alternative for viruscell fusion applications.

Applications: Applications of pyrene excimer assays for membrane fusion are described in the footnoted references.26,28,4043

NOTE 13.1

Lipid-Mixing Assays of Membrane Fusion







1. Chem Phys Lipids (2002) 116:39; 2. Anal Biochem (2009) 386:91; 3. Methods Enzymol (1993) 220:3; 4. Methods Enzymol (1993) 220:15; 5. Proc Natl Acad Sci U S A (2009) 106:979; 6. Annu Rev Biophys Biophys Chem (1989) 18:187; 7. Hepatology (1990) 12:61S-66S; 8. Biochemistry (1987) 26:8435; 9. Biochemistry (1981) 20:4093; 10. Methods Enzymol (1993) 221:239; 11. Biochemistry (1994) 33:12615; 12. Biochemistry (1994) 33:5805; 13. Biochemistry (1994) 33:3201; 14. Biophys J (1994) 67:1117; 15. J Biol Chem (1994) 269:15124; 16. J Biol Chem (1994) 269:4050; 17. J Biol Chem (1993) 268:1716; 18. Biochemistry (1992) 31:2629; 19. Biochemistry (1991) 30:5319; 20. J Biol Chem (1991) 266:3252; 21. Biochemistry (1984) 23:5675; 22. J Biol Chem (1990) 265:13533; 23. Biophys J (1993) 65:325; 24. Biophys J (1990) 58:1157; 25. Biochim Biophys Acta (1994) 1190:360; 26. Biochemistry (1993) 32:11330; 27. Biochemistry (1993) 32:900; 28. Biochemistry (1994) 33:9110; 29. Biochemistry (1994) 33:1977; 30. Biochim Biophys Acta (1994) 1191:375; 31. J Biol Chem (1994) 269:5467; 32. Biochem J (1993) 294:325; 33. J Biol Chem (1993) 268:25764; 34. J Biol Chem (1993) 268:9267; 35. Virology (1993) 195:855; 36. Biochemistry (1992) 31:10108; 37. Exp Cell Res (1991) 195:137; 38. J Virol (1991) 65:4063; 39. Biochemistry (1990) 29:4054; 40. EMBO J (1993) 12:693; 41. J Virol (1992) 66:7309; 42. Biochemistry (1988) 27:30; 43. Biochim Biophys Acta (1986) 860:301.

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Wavelength (nm)350 450400 500 550 600

1

2

3

4

Figure 3 Excimer formation by pyrene in ethanol. Spectra are normalized to the 371.5 nm peak of the monomer. All spectra are essentially identical below 400 nm after normalization. Spectra are as follows: 1) 2 mM pyrene, purged with argon to remove oxygen; 2) 2 mM pyrene, air-equilibrated; 3) 0.5 mM pyrene (argon-purged); and 4) 2 M pyrene (argon-purged). The monomer-to-excimer ratio (371.5 nm/470 nm) is dependent on both pyrene concentration and the excited-state lifetime, which is variable because of quenching by oxygen.

Selected applications of cis-parinaric acid include:

Measurement of peroxidation in lipoproteins 6365 and the relationship of peroxidation to cytotoxicity 66,67 and apoptosis 6871

Evaluation of antioxidants 7275

Detection of lipoproteins following chromatographic separation 76 and structural charac-terization of lipoproteins 77

Detection of lipidprotein interactions 7880 and lipid clustering 81

High-anity binding to a hydrophobic pocket between the heavy chain of myosin subfrag-ment-1 and its essential light chain 82

Investigation of the mechanism of fatty acidbinding proteins 8385 and phospholipid-trans-fer proteins 86,87

e extensive unsaturation of cis-parinaric acid makes it quite susceptible to oxida-tion. Consequently, we oer cis-parinaric acid in a 10 mL unit size of a 3 mM solution in de-oxygenated ethanol (P36005); if stored protected from light under an inert argon atmosphere at 20C, this stock solution should be stable for at least six months. During experiments, we strongly advise handling cis-parinaric acid samples under inert gas and preparing solutions us-ing degassed buers and solvents. cis-Parinaric acid is also somewhat photolabile, undergoing photodimerization under intense illumination, resulting in loss of uorescence.88

ADIFAB Fatty Acid IndicatorFatty acidbinding proteins are small cytosolic proteins found in a variety of mamma-

lian tissues, and studies of their physiological function frequently involve uorescent fatty acid probes.89 To facilitate these studies, we oer ADIFAB reagent (A3880), a dual-wavelength uo-rescent indicator of free fatty acids 9092 (Figure 13.2.13, Figure 13.2.14). ADIFAB reagent is a conjugate of I-FABP, a rat intestinal fatty acidbinding protein with a low molecular weight (15,000 daltons) and a high binding anity for free fatty acids,93 and the polarity-sensitive acrylodan uorophore (A433, Section 2.3). It is designed to provide quantitative monitoring of free fatty acids without resorting to separative biochemical methods.44,94,95 With appropriate precautions, which are described in the product information sheet accompanying this product, ADIFAB can be used to determine free fatty acid concentrations between 1 nM and >20 M.

Figure 13.2.13 Ribbon representation of the ADIFAB free fatty acid indicator (A3880). In the left-hand image, the fatty acid binding site of intestinal fatty acidbinding protein (yellow) is occupied by a cova-lently attached acrylodan uorophore (blue). In the right-hand image, a fatty acid molecule (gray) binds to the protein, displacing the uoro-phore (green) and producing a shift of its uorescence emission spec-trum. Image contributed by Alan Kleinfeld, FFA Sciences LLC, San Diego.

Figure 13.2.14 The free fatty aciddependent spectral shift of ADIFAB (A3880). Spectra shown represent 0.2 M ADIFAB in pH 8.0 buer with (+OA) and without (OA) addition of 4.7 M cis-9-octadecenoic (oleic) acid (OA). The ratio of uorescence emission intensities at 505 nm and 432 nm can be quantitatively related to free fatty acid concentrations.

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_OA

+OA

400 450 500 550 600 650

Wavelength (nm)

Ex = 390 nm








Phospholipids with BODIPY DyeLabeled Acyl ChainsBODIPY Glycerophospholipids

We oer several glycerophospholipid analogs labeled with a single green-uorescent BODIPY 500/510 or a BODIPY FL uorophore or red-orangeuorescent BODIPY 530/550 uorophore on the sn-2 acyl chain, including:

BODIPY 500/510 dyelabeled glycerophosphocholine (D3793, Figure 13.2.15)

BODIPY FL dyelabeled glycerophosphocholine (D3792, Figure 13.2.16; D3803, Figure 13.2.17)

BODIPY 530/550 dyelabeled glycerophosphocholine (D3815, Figure 13.2.18)

BODIPY FL dyelabeled phosphatidic acid (D3805, Figure 13.2.19)

In addition, we prepare a glycerophosphocholine analog with a single nonhydrolyzable ether-linked BODIPY FL uorophore on the sn-1 position (D3771, Figure 13.2.20), as well as several doubly labeled glycerophospholip-ids. ese doubly labeled glycerophospholipids, which are discussed in greater detail in Section 17.4, are designed primarily for detection of phospholipase A1 and phospholipase A2 and include:

Glycerophosphoethanolamine with a BODIPY FL dyelabeled sn-1 acyl chain and a dinitrophenyl quenchermodied headgroup (PED-A1, A10070; Figure 13.2.21)

Glycerophosphoethanolamine with a BODIPY FL dyelabeled sn-2 acyl chain and a dinitrophenyl quenchermodied headgroup 96 (PED6, D23739; Figure 13.2.22)

Glycerophosphocholine with two BODIPY FL dyelabeled acyl chains (bis-BODIPY FL C11-PC, B7701; Figure 13.2.22)

Glycerophosphocholine with a BODIPY 558/568 dyelabeled sn-1 alkyl chain and a BODIPY FL dyelabeled sn-2 acyl chain (Red/Green BODIPY PC-A2, A10072; Figure 13.2.23)

e spectral properties of BODIPY FL dyelabeled phospholipids are summarized in Table 13.2. Unlike the nitrobenzoxadiazole (NBD) uorophore, the BODIPY FL and BODIPY 500/510 uorophores are intrinsically lipo-philic and readily localize in the membranes interior.1 e uorophore is com-pletely inaccessible to the membrane-impermeant antiBODIPY FL antibody (A5770, Section 7.4), which also recognizes the BODIPY 500/510 derivative. As shown in Figure 13.2.24, the emission spectrum of the BODIPY 500/510 uo-rophore is much narrower than that of the NBD uorophore. Because both the extinction coecient of the BODIPY 500/510 uorophore and its quantum yield in a lipophilic environment (EC ~90,000 cm1M1 and QY ~0.9) are much higher than those of the NBD uorophore (EC ~20,000 cm1M1 and QY ~0.3), much less BODIPY 500/510 dyelabeled phospholipid is required for labeling membranes.4

C OCH2

OCH

CH2O OCH2CH2N(CH3)3

O

O

CH3(CH2)1

O

FFN

BN

(CH2)11

H3C

H3C CO

C OCH2

OCH

CH2O OCH2CH2N(CH3)3

O

O

CH3(CH2)1

O

FFN

BN

(CH2)

H3C

H3C CO

C OCH2

OCH

CH2O OCH2CH2N(CH3)3

O

O

CH3(CH2)1

O

FFN

BN

(CH2) CO

Figure 13.2.15 2-(4,4-Diuoro-5-methyl-4-bora-3a,4a-diaza-s-indacene-3-dodecano-yl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (-BODIPY 500/510 C12-HPC, D3793).

Figure 13.2.16 2-(4,4-Diuoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (-BODIPY FL C12-HPC, D3792).

Figure 13.2.17 2-(4,4-Diuoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pen-tanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (-BODIPY FL C5-HPC, D3803).

Figure 13.2.18 2-(4,4-Diuoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-pen-tanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine (-BODIPY 530/550 C5-HPC, D3815).

Figure 13.2.19 2-(4,4-Diuoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pen-tanoyl)-1-hexadecanoyl-sn-glycero-3-phosphate, diammonium salt (-BODIPY FL C5-HPA, D3805).

C OCH2

OCH

CH2O OO

O

CH3(CH2)1

O

FFN

BN

(CH2)

H3C

H3C CO 2NH

Figure 13.2.21 PED-A1 (N-((6-(2,4-DNP)amino)hexanoyl)-1-(BODIPY FL C5)-2-hexyl-sn-glycero-3-phosphoethanolamine; A10070).

C OCH2

CH3(CH2)5 OCH

CH2O OCH2CH2NHO

OH

(CH2)

O

C (CH2)5NHO

O2N

NO2

FFH3C

NB

N

H3C

Figure 13.2.20 2-Decanoyl-1-(O-(11-(4,4-diuoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl)amino)undecyl)-sn-glycero-3-phosphocholine (D3771).







Figure 13.2.23 Red/Green BODIPY PC-A2 (1-O-(6-BODIPY 558/568-aminohexyl)-2-BODIPY FL C5-sn-glycero-3-phosphocholine; A10072).

FFN

BN

CH2CH2FF

NB

N

(CH2) C OCHO

CH2O OCH2CH2N(CH3)3

O

O

H3C

H3C

C NH(CH2)

OOCH2

Figure 13.2.22 Mechanism of phospholipase activitylinked uorescence enhancement responses of bis-BODIPY FL C11-PC (B7701) and PED6 (D23739). Note that enzymatic cleavage of bis-BODIPY FL C11-PC yields two uorescent products, whereas cleavage of PED6 yields only one.

+

Fluorescent lysophospholipid

Quenched substrate

+

C

O

(CH2)10H3C

H3C

F F

NB

N

C

O

(CH2)10H3C

H3C

F F

NB

N CH2O

OCH

OCH2

OCH2CH2N(CH3)3O

O

P

(bis-BODIPY FL C11-PC)

Fluorescent fatty acid (BODIPY FL C11 (D3862))

Phospholipase A2

C

O

(CH2)10H3C

H3C

F F

NB

N

OH

+P

O

O

OCH2CH2N(CH3)3

OCH2HOCH

CH2O

NB

N

FF

H3C

H3C (CH2)10

O

C

NB

N

F F

+

Nonuorescent lysophospholipid

Quenched substrate (PED6)

NO2

O2N

(CH2)5NH

O

COCH2CH2NH

O

O

P

CH3(CH2)14

O

C

CH2O

OCH

OCH2C

O

(CH2)4H3C

H3C

CH3(CH2)14

O

C OCH2HOCH

CH2O P

O

O

OCH2CH2NH C

O

(CH2)5NH

O2N

NO2

Phospholipase A2

Fluorescent fatty acid (BODIPY FL C5 (D3834))

C

O

(CH2)4H3C

H3C

F F

NB

N

OH

Table 13.2 Spectral properties of some lipid probes.

Spectral Property Pyrene DPH NBD BODIPY FL

Ex/Em (nm)* 340/376 360/430 470/530 507/513

QY () 0.6 (>100 nanoseconds) 0.8 (48 nanoseconds) 0.32 (510 nanoseconds) 0.9 (6 nanoseconds)

Concentration dependence Excimer emission (~470 nm) at high concentrations.

Self-quenched at high concentrations.

Self-quenched at high concentrations.

Excimer emission (~620 nm) at high concentrations.

Environmental sensitivity Very sensitive to quenching by oxygen. Essentially nonuorescent in water.

Essentially nonuorescent in water.

Essentially nonuorescent in water.

Relatively insensitive. Strongly uorescent in both aqueous and lipid environments.

*Typical uorescence excitation and emission maxima for membrane-intercalated probes. QY = uorescence quantum yield; = uorescence decay lifetime. Typical values for membrane-intercalated probes are listed. These values may show signicant environment-dependent variations.

Figure 13.2.24 Fluorescence spectra (exci-tation at 475 nm) of -BODIPY 500/510 C12-HPC (blue line peak at 516 nm, D3793) and NBD C12-HPC (red line peak at 545 nm, N3787) incorporated in DOPC (dioctadecenoylg-lycerophosphocholine) liposomes at molar ratios of 1:400 mole:mole (labeled:unlabeled PC). The integrated intensities of the spectra are proportional to the relative uorescence quantum yields of the two probes.

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Wavelength (nm)

BODIPY-PC (D-3793)

NBD-PC (N-3787)

500 550 600 650








Incorporation of high molar ratios (>10 mole %) of the BODIPY 500/510 dyelabeled phospholipids into membranes results in a dramatic spectral shi of the uorescence emission spectrum to longer wavelengths (Figure 13.2.25). We have also observed this spectral shi in the Golgi of live cells that have been labeled with our BODIPY dyelabeled ceramides (Section 12.4) and with BODIPY fatty acids that have been metabolically incorporated by cells (Figure 13.2.2). In uorescence resonance energy transfer (FRET) measurements, the green-uorescent BODIPY 500/510 dye is an excellent donor to longer-wavelength BODIPY probes 97,98 (Figure 13.2.26) and acceptor from dansyl-, DPH- or pyrene-labeled phospholipids.99 ese probe combinations oer several alternatives to the widely used NBDrhodamine uorophore pair for researchers using FRET techniques to study lipid transfer and membrane fusion.97

ApplicationsOnce cells are labeled with a BODIPY phospholipid, the probe

shows little tendency to spontaneously transfer between cells.100 Consequently, BODIPY dyelabeled phospholipids have been used in a number of studies of cell membrane structure and properties:

Despite their good photostability, BODIPY lipids are useful for uorescence recovery aer photobleaching (FRAP) measurements of lipid diusion.101,102

Researchers have used BODIPY fatty acids and phospholip-ids to visualize compartmentalization of specic lipid classes in Schistosoma mansoni 103 and fungi.104,105

-BODIPY FL C12-HPC (D3792) has been used to examine lip-idprotein interactions involved in bacterial protein secretion via uorescence resonance energy transfer (FRET) measurements 106 (Fluorescence Resonance Energy Transfer (FRET)Note 1.2).

-BODIPY FL C5-HPC 107 (D3803) has been used to character-ize lipid domains by uorescence correlation spectroscopy 108 (Fluorescence Correlation Spectroscopy (FCS)Note 1.3), confo-cal laser-scanning microscopy 109 (Figure 13.2.27) and near-eld scanning optical microscopy.101,110

bis-BODIPY FL C11-PC (B7701) has BODIPY FL dyelabeled sn-1 and sn-2 acyl groups (Figure 13.2.28), resulting in partially quenched uorescence that increases when one of the acyl groups is hydrolyzed by phospholipase A1 or A2. e hydrolysis products are BODIPY FL undecanoic acid (D3862) and BODIPY FL dyela-beled lysophosphatidylcholine (Figure 13.2.22). e probe has been used successfully in human neutrophils, plants and zebrash to detect phospholipase A activity 111116 (Section 17.4).

-BODIPY FL C5-HPC (D3803) has been used to investigate the cellular uptake of antineoplastic ether lipids.117

Figure 13.2.25 A) Fluorescence spectrum of -C8-BODIPY 500/510 C5-HPC (D3795) incorporated in DOPC (dioctadecenoylphosphocholine) liposomes at 1:100 mole:mole (labeled:unlabeled PC). B) Fluorescence spectra at high molar incorporation levels: 1:10 mole:mole and 1:5 mole:mole.

A

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Wavelength (nm)500 600 700

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Wavelength (nm)500 600 700

B1:10

1:5

Figure 13.2.26 Fluorescence resonance energy transfer from -BODIPY 500/510 C12-HPC (peak at 516 nm, D3793) to BODIPY 558/568 C12 (peak at 572 nm, D3835) in DOPC (diocta-decenoylglycerophosphocholine) lipid bilayers using 475 nm excitation. Ratio of acceptors to donors is: 1) 0; 2) 0.2; 3) 0.4; 4) 0.8; and 5) 2.0.

1

2

3

4

5

5

4

3

2

1

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Wavelength (nm)500 550 600 650

Figure 13.2.27 Confocal laser-scanning microscopy images of a giant unilamellar phospho-lipid vesicle (GUV). The lipid composition of this GUV was DPPC/DLPC = 1/1, with DiIC20(3) and -BODIPY FL C5-HPC (D3803) dyes at mole fraction ~0.001. Excitation was at 488 nm. The upper left image is the uorescence emission through a 585 nm longpass lter, thus almost exclusively from DiIC20(3). The lower left image is the emission through a 522 35 nm band-pass lter, thus almost exclusively from -BODIPY FL C5-HPC. The right image is color-merged, using the public domain NIH Image program. Image contributed by Gerald W. Feigenson, Cornell University, and reprinted with permission from Biophys J (2001) 80:2775.

Figure 13.2.28 1,2-bis-(4,4-diuoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-undecanoyl)-sn-glycero-3-phosphocholine (bis-BODIPY FL C11-PC, B7701).







Phospholipid with DPH-Labeled Acyl ChainProperties

e uorescent phospholipid analog -DPH HPC (D476) comprises diphenylhexatriene propionic acid coupled to glycerophosphocholine at the sn-2 position (Figure 13.2.29). It is therefore related to the neutral membrane probe DPH and the cationic derivative TMA-DPH (D202, T204; Section 13.5). DPH and its derivatives exhibit strong uorescence enhancement when incorporated into membranes, as well as sensi-tive uorescence polarization (anisotropy) responses to lipid ordering (Fluorescence Polarization (FP)Note 1.4). -DPH HPC was originally devised to improve the localization of DPH in membranes.118,119 Unlike underivatized DPH, it can be used to specically label one leaet of a lipid bilayer, facilitating analysis of membrane asymmetry.120

ApplicationsDPH derivatives are predominantly used to investigate the struc-

ture and dynamics of the membrane interior either by uorescence polarization or lifetime measurements. Researchers have used -DPH HPC as a probe for lipidprotein interactions,121123 alcohol-induced perturbations of membrane structure,124,125 molecular organiza-tion and dynamics of lipid bilayers 11,126128 and lipid peroxidation.129 Fluorescence lifetime measurements of -DPH HPC provide a sen-sitive indicator of membrane fusion.130132 In addition to membrane fusion, -DPH HPC has been used to monitor various other lipid-transfer processes.133135

Phospholipids with NBD-Labeled Acyl ChainsProperties

Our acyl-modied nitrobenzoxadiazole (NBD) phospholipid probes include both the NBD hexanoyl- and NBD dodecanoyl-glyc-erophosphocholines (NBD C6-HPC, N3786; Figure 13.2.30 and NBD C12-HPC, N3787). Table 13.2 compares the spectral properties of these probes with those of the BODIPY, DPH and pyrene lipid probes. Unlike the BODIPY phospholipids, the location of the relatively polar NBD uorophore of NBD C6-HPC and NBD C12-HPC in phospholipid bi-layers does not appear to conform to expectations based on the probe structure. A variety of physical evidence indicates that the NBD moiety "loops back" to the head-group region 136139 (Figure 13.2.1). In fact, the uorophore in this acyl-modied phospholipid appears to probe the same location as does the head grouplabeled glycerophosphoethanol-amine derivative NBD-PE 17 (N360).

ese NBD probes transfer spontaneously between membranes, with NBD C6-HPC transferring more rapidly than its more lipo-philic C12 analog.140,141 NBD C6-HPC can be readily removed (back-exchanged) from the plasma membrane by incubating the labeled cells either with unlabeled lipid vesicles 142 or with bovine serum al-bumin.143145 is property is useful for quantitating lipid transfer and for studying phospholipid distribution asymmetry and transmembrane "ip-op" rates in lipid bilayers.17,146151

Figure 13.2.29 -DPH HPC (2-(3-(diphenylhexatrienyl)propanoyl)-1-hexadecanoyl-sn-glycero-3-phosphocholine; D476).

Figure 13.2.30 2-(6-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine (NBD C6-HPC, N3786).

ApplicationsNBD acylmodied probes are used for investigating lipid traf-

c, either by directly visualizing NBD uorescence,152155 by exploit-ing NBD self-quenching 156158 or by uorescence resonance energy transfer methods.140,152,159161 Lateral domains in model monolayers, bilayers and cell membranes have been characterized using NBD phos-pholipids in conjunction with uorescence recovery aer photobleach-ing 162164 (FRAP), uorescence resonance energy transfer 165 (FRET) (Fluorescence Resonance Energy Transfer (FRET)Note 1.2) and di-rect microscopy techniques.166169 Transmembrane lipid distribution (Lipid-Mixing Assays of Membrane FusionNote 13.1) has been as-sessed using uorescence resonance energy transfer from NBD HPC to rhodamine DHPE 149,151,170 (L1392) or alternatively by selective dithi-onite (S2O42) reduction of NBD phospholipids in the outer membrane monolayer 171 (Figure 13.2.31).

Figure 13.2.31 Dithionite reduction of 6-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexa-noic acid (NBD-X, N316). The elimination of uorescence associated with this reaction, coupled with the fact that extraneously added dithionite is not membrane permeant, can be used to determine whether the NBD uorophore is located in the external or internal monolayer of lipid bilayer membranes.

N

O

N

NO2

NH(CH2)5 C

O

OH

N

O

N

NH2

NH(CH2)5 C

O

OH

S2O42

Fluorescent Nonuorescent








Phospholipids with Pyrene-Labeled Acyl ChainsProperties

Phospholipid analogs with pyrene-labeled sn-2 acyl chains (Figure 13.2.32) are among the most popular uorescent membrane probes.29,172,173 We oer pyrenedecanoyl-labeled glycerophospholipids with phosphocholine (H361) and phosphoglycerol (H3809) head groups.

e spectral properties of the pyrene lipid probes are summarized in Table 13.2. Of primary importance in terms of practical applications is the concentration-dependent for-mation of excited-state pyrene dimers (excimers), which exhibit a distinctive red-shied emission (peak ~470 nm) (Figure 13.2.10).

Applicationse excimer-forming properties of pyrene are well suited for monitoring membrane

fusion (Lipid-Mixing Assays of Membrane FusionNote 13.1) and phospholipid transfer processes.37,173178 e monomer/excimer emission ratio can also be used to characterize membrane structural domains and their dependence on temperature, lipid composition and other external factors.179182 Pyrenedecanoyl glycerophosphocholine (-py-C10-HPC, H361) has been used to elucidate the eect of extrinsic species such as Ca2+,183 platelet-activating factor,184 drugs,185 membrane-associated proteins 186188 and ethanol 189 on lipid bilayer structure and dynamics. e anionic phosphoglycerol analog (H3809, Figure 13.2.33) is preferred as a substrate for secretory phosholipases A2 relative to other phos-pholipid classes.190,191 e long excited-state lifetime of pyrene (Table 13.2) renders the uorescence of its conjugates very susceptible to oxygen quenching, and consequently these probes can be used to measure oxygen concentrations in solutions,192 lipid bilay-ers 193 and cells.194,195

Glycerophospholipids in which both alcohols are esteried to pyrene fatty acids (Figure 13.2.1), as in our bis-(1-pyrenebutanoyl)- and bis-(1-pyrenedecanoyl)glycero-phosphocholines (B3781, Figure 13.2.34; B3782) show strong excimer uorescence, with maximum emission near 470 nm.29 Hydrolysis of either fatty acid ester by a phospholipase results in liberation of a pyrene fatty acid and an emission shi to shorter wavelengths, making these probes useful as phospholipase substrates 196199 (Section 17.4).

For detecting labels at the membrane surface, we oer antibodies that recognize the following labels:

Alexa Fluor 488 dye (A11094) BODIPY FL dye (A5770) Alexa Fluor 405 and Cascade Blue dyes (A5760) Dansyl (A6398) Dinitrophenyl chromophore (A6423, A6430, A6435, A11097,

Q17421MP) Fluorescein and Oregon Green dyes (A889, A982, A6413, A6421,

A11090, A11091, A11095, A11096, Q15421MP, Q15431MP) Green Fluorescent Protein (GFP, A6455, A10259, A10262, A10263,

A11120, A11121, A11122, A21311, A21312, A31851, A31852, G10362)

Lucifer yellow (A5750, A5751) Tetramethylrhodamine (A6397) Texas Red dye (A6399)

NOTE 13.2

Antibodies for Detecting Membrane-Surface Labels

Figure 13.2.32 1-Hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine (-py-C10-HPC, H361).

Figure 13.2.33 1-Hexadecanoyl-2-(1-pyrenedecanoyl)-sn-glycero-3-phosphoglycerol, ammonium salt (-py-C10-PG, H3809).

Figure 13.2.34 1,2-Bis-(1-pyrenebutanoyl)-sn-glycero-3-phosphocholine (B3781).

Fluorescent conjugates of several of these anti-dye and anti-hapten antibodies are available; see Section 7.4 and Table 7.8 for complete product information. These antibodies can be used for direct detection of labeled phospholipids via uorescence quenching1,2 (or uorescence enhancement, in the case of the anti-dansyl antibody). When used in conjunction with phospho-lipids with dye-labeled head groups (Table 13.1), they are important tools for:

Studies of molecular recognition mechanisms at membrane surfaces3

Lipid diusion measurements4,5

Quantitation of lipid internalization by endocytosis6,7

In addition to anti-fluorophore antibodies, we offer a selection of streptavidin conjugates (Section 7.6, Table 7.9) for detecting biotinylated phospholipids.

1. Biochemistry (1999) 38:976; 2. J Biol Chem (1998) 273:22950; 3. Angew Chem Int Ed Engl (1990) 29:1269; 4. J Cell Biol (1993) 120:25; 5. Proc Natl Acad Sci U S A (1991) 88:6274; 6. J Cell Biol (1988) 106:1083; 7. Cell (1991) 64:393.







Phospholipids with a Fluorescent or Biotinylated Head Group

Phospholipid with a Dansyl-Labeled Head Groupe phospholipid analog incorporating the environment-sen-

sitive 200 dansyl uorophore (dansyl DHPE, D57; Figure 13.2.35) is a useful probe of lipidwater interfaces.53,201 It is sensitive to the interac-tions of a number of proteins, including protein kinase C,202,203 annex-ins 204,205 and phospholipase A2,206208 with membrane surfaces. Dansyl DHPE has also been used to examine the eects of cholesterol on the accessibility of the dansyl hapten to antibodies 209 (Antibodies for Detecting Membrane-Surface LabelsNote 13.2).

Phospholipid with a Marina Blue DyeLabeled Head Group

Marina Blue DHPE (M12652, Figure 13.2.36) is optimally excited by the intense 365 nm spectral line of the mercury-arc lamp and ex-hibits bright blue uorescence emission near 460 nm. Signicantly, the pKa value of this 6,8-diuoro-7-hydroxycoumarin derivative is 23 log units lower than that of nonuorinated 7-hydroxycoumarin analogs; consequently, Marina Blue DHPE is expected to be strongly uorescent in membranes, even at neutral pH.

Phospholipid with a Pacic Blue DyeLabeled Head Group

e Pacic Blue dyelabeled phospholipid (Pacic Blue DMPE, P22652; Figure 13.2.37) is our only head grouplabeled phospholipid with tetradecanoyl (myristoyl) esters rather than hexadecanoyl (pal-mitoyl) esters. is blue-uorescent phospholipid is structurally similar to a phospholipid described by Gonzalez and Tsien for use in a FRET-based measurement of membrane potential.210

Figure 13.2.35 N-(5-dimethylaminonaphthalene-1-sulfonyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (dansyl DHPE, D57).

C OCH2

C OCH

CH2O OCH2CH2NHO

O

CH3(CH2)1

O

CH3(CH2)1O

(CH3CH2)3NH N(CH3)2

O2

Figure 13.2.36 Marina Blue 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (Marina Blue DHPE, M12652).

Figure 13.2.37 Pacic Blue DMPE (Pacic Blue 1,2-ditetradecanoyl-sn-glycero-3-phos-phoethanolamine, triethylammonium salt; P22652).

C OCH2

C OCH

CH2O OCH2CH2NHO

O

CH3(CH2)12

O

CH3(CH2)12O

(CH3CH2)3NH

OF

F

OH

CO

O

Figure 13.2.38 NBD-PE (N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt; N360).

Phospholipid with an NBD-Labeled Head Groupe widely used membrane probe nitrobenzoxadiazolyldihexadecan-

oylglycerophosphoethanolamine 17 (NBD-PE, N360; Figure 13.2.38) has three important optical properties: photolability, which makes it suitable for photobleaching recovery measurements; concentration-dependent self-quenching; and uorescence resonance energy transfer to rhodamine acceptors (usually rhodamine DHPE, L1392). Spectroscopic characteris-tics of NBD-PE are generally similar to those described for our phospho-lipids with NBD-labeled acyl chains (N3786, N3787). NBD-PE is frequent-ly used in NBDrhodamine uorescence energy transfer experiments to monitor membrane fusion (Lipid-Mixing Assays of Membrane FusionNote 13.1). In addition, this method can be used to detect lipid domain formation 165 and intermembrane lipid transfer 211214 and to determine the transbilayer distribution of phospholipids.151 Attachment of the NBD uo-rophore to the head group makes NBD-PE resistant to transfer between vesicles.142 NBD-PE has been used in combination with either rhodamine DHPE (L1392) or Texas Red DHPE (T1395MP) for visualizing the spa-tial relationships of lipid populations by uorescence resonance energy transfer microscopy.215 e nitro group of NBD can be reduced with so-dium dithionite, irreversibly eliminating the dyes uorescence (Figure 13.2.31). is technique can be employed to determine whether the probe is localized on the outer or inner leaet of the cell membrane.171,216218 e argon-ion laserexcitable NBD-PE is also a frequent choice for uores-cence recovery aer photobleaching (FRAP) measurements of lateral dif-fusion in membranes.219222 In addition, NBD-PE is of particular value for monitoring bilayer-to-hexagonal phase transitions, because these transi-tions cause an increase in NBD-PEs uorescence intensity.223225

Phospholipid with a Fluorescein-Labeled Head GroupFluorescein-derivatized dihexadecanoylglycerophosphoetha-

nolamine (uorescein DHPE, F362; Figure 13.2.39) is a membrane-surface probe that is sensitive to both the local electrostatic potential and pH.226228 An antiuorescein/Oregon Green antibody (A889, Section7.4) has been employed in combination with uorescein DHPE

Figure 13.2.39 Fluorescein DHPE (N-(uorescein-5-thiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-pho

The Molecular Probes® Handbook A GUIDE TO ... 13 Probes for Lipids and Membranes Molecular Probes Handbook A Guide to Fluorescent Probes and Labeling Technologies 11th Edition (2010)

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