Thuesday: MACS ® Technology for magnetic isolation of cells and molecules Introduction Features of paramagnetic MicroBeads General procedure of magnetic.

Thuesday:MACS® Technology for magnetic isolation of cells and molecules

• Introduction • Features of paramagnetic MicroBeads • General procedure of magnetic cell isolation • Overview of applications in molecular biology

µMACS epitope tagged protein isolation • Expression of tagged/fusion proteins, e.g. GFP fusion proteins • Magnetic protein isolation

Wednesday: • Detection of proteins, results and trouble-shooting • Optimizing protein expression analysis by transfected cell selection (MACSelect)

• organism for protein expression (bacteria, eukaryotic)

• expression vector (promoter etc.)

• transient or stable transfection

• choice of tag

Expression of tagged/fusion proteins, e.g. GFP fusion proteins

General features of the MicroBeads

• Super-paramagnetic

• Colloidal non-sedimenting Extremly fast reactionkinetics & efficient

• Extremely small magnetic labeling

• Biodegradable & non-toxic

Magnetic protein isolation

1. Cell lysis

2. Magnetic labeling with MicroBeads

• µMACS Protein A and Protein G MicroBeads

• µMACS Anti Epitope Tag MicroBeads, e.g. Anti-GFP

MicroBeads

• µMACS Streptavidin MicroBeads

3. Specific protein isolation with µ or M Columns and MACS® Separator

Immunoprecipitation with µMACSTM Protein A / Protein G MicroBeads

Cell lysis andimmunolabeling

Magneticseparation

SDS-PAGE orenzymatic assay

Pure protein in less than 2 hours

Immunoprecipitation with µMACS

Silver stained SDS PAGE

L: Lysate

M: Marker

Lane 1: Large T antigen (indicated by arrow)

Lane 2: Isotype control

Lane 3: Negative control

IP of SV40 large T antigen from COS cells with µMACSTM Protein G MicroBeads

Immunoprecipitation of surface antigens

IP of biotinylated surface antigens of B cell line JOK-1 with µMACSTM Protein A

Western Blot with Streptavidin-HRP

Lane 2: anti-CD22 (HD 239)

Lane 4: anti-MHC class I (W6/32)

Lane 1 and 3: isotype matched antibody as control.

1 2 3 4

CD 22 (140 kDa)

MHC Class I (45 kDa)

Co-IP of the androgen receptor and it´s target

Western Blot of Immunoprecipitated AR using Anti-AR antibody

Western Blot of AR-co-immunoprecipitated Beta-Catenin using anti-Beta-Catenin antibody

AR was immunoprecipitaed from DHT-stimulated (lane 1, 3) or unstimulated LNCaP cells (lane 2, 4) with µMACS Protein G MicroBeads (lane 1-2) or with Protein A/G agarose beads (lane 3-4).

Courtesy of D. Mulholland, Vancouver, Canada

ChIP - Chromatin immunoprecipitation

PCR of the PSA gene using ChIP obtained DNA

IP was carried with µMACS Protein G MicroBeads (lane 1-2) or with Protein A/G agarose beads (lane 3-4). Anti-AR antibody (lane 1, 3) or as a control anti-IgG (lane 2, 4) were used for Chip.

Courtesy of D. Mulholland, Vancouver, Canada

µMACSTM Epitope Tag Protein Isolation Kits

Application

isolation of recombinant proteins with epitope tags

and their interaction partners from eukaryotic cells

µMACSTM Epitope Tag Protein Isolation Kits

Pure proteins in less than 2 hours

• highly specific MicroBeads coupled to monoclonal

antibodies

• eliminate background with effective column washing

• optimized buffer set, small elution volume

• MACS® Columns for easy handling

Epitope-tags

• His : artificial tag of 6 Histidines

• HA= Hemaglutinin (or Hemagglutinin) : viral gene

• c-myc : human c-myc is proto-oncogene

• GST = Glutathion S-transferase

• GFP = Green Fluorescent Protein

BM 10/2002

µMACS Epitope Tagged Protein Isolation Kits

GFP = Green Fluorescent Protein from Aequoria victoria

BM 10/2002

Zur Anzeige wird der QuickTime™ Dekompressor “GIF”

benötigt.

MACS in Molecular Biology

MACS in Molecular Biology

Epitope tagged proteins - Advantages:

• study of proteins possible without a specific antibody (production of specific Ab difficult & time-consuming)

• simplified isolation: known epitope + known interaction

• standardized isolation: using one established system (expression vector/tag) for study of different proteins

BM 10/2002

Standardized isolation of different proteins

• recombinant proteins A-C isolated with protein specific Ab

BM 10/2002

Zur Anzeige wird der QuickTime™ Dekompressor “GIF”

benötigt.

• tagged recombinant proteins A-C isolated with tag specific Ab

µMACS™ Epitope Tagged Protein Isolation Kits

MACS in Molecular Biology

Epitope tagged proteins: working areas - examples

• DNA sequences (e.g. identified by HUGO =human genome organization) give no information about

• structure

• binding partners

• function

• cellular localisation

BM 10/2002

they need to be expressed in eukaryotic cells

Working scheme for isolation of tagged proteins

Lyse cells and add MicroBeads

Apply to MACS Column

Remove unbound material by stringent washing

Elute target protein while MicroBeads remain on column

µMACSTM Epitope Tag Protein Isolation Kits

Protein isolation with µMACSTM Anti-c-myc MicroBeads

Western Blot anti-c-myc/POD

Lane 1 -3: 10, 20, 50 ng of Multi-Tag Protein

Lane 4: c-myc-BDCA2 (1/25 eluat)

Lane 5:c-myc-Dectin2 (1/125 eluat)

Lane 6: c-myc-BDCA2 (1/50 eluat)

Lane 7: c-myc- Dectin2 (1/250 eluat)

Cell source: 5x 10E6 293 T HEK (human embryonal kidney)

Isolation of c-myc-BDCA2 from biotinylated cell lysate

1 2 3 4 5 6 7

40 kDa -

30 kDa -

24 kDa -

Isolation c-myc-BDCA-2 using anti-HA and anti-c-myc MicroBeads

© Miltenyi Biotec

c-myc-

BDCA-2 a

nti-

HA

ant

i-c-

myc

35S-methionine labeled isolation

Application

isolation of any biotinylated molecules like DNA,

RNA, proteins, etc. and their interacting molecules

µMACSTM Streptavidin Kit

• Isolation of DNA-binding proteins (transcription studies)

• Isolation of RNA-binding proteins (translation studies)

• Isolation of (specific) transcripts (e.g. viral transcripts)

• Phage display

• Subtractive hybridisation

• SAGE

• Isolation of tRNA molecules

µMACSTM Strepavidin Kit

Isolation of target molecules with biotinylated probes, here the isolation of DNA-binding protein. Incubate your sample of interest with biotinylated DNA probe (1), add µMACS Streptavidin MicroBeads (2) and apply to column (4). Wash to remove unbound material (5) and elute your target molecule while the biotinylated probe remains on the column (6).

Isolation of DNA-binding proteins

1 2 3 4 5 6

Transcription control studies with µMACSTM Streptavidin Kit

Purification of DNA binding proteins

IL-4 nonMarker stimulated stimulated

Stat6

Western blot of Stat6 transcription factor. Dermal fibroblasts were stimulated with IL-4 and their nucleus extracts were incubated with biotinylated DNA already coupled to µMACS Streptavidin MicroBeads. The isolated binding proteins were purified and proofed by SDS-PAGE and Western blotting

Isolation of a DNA binding protein

Using biotinylated target DNA for LNCaP nuclear protein associated with PSA promotor region

2D gel electrophoresis of protein bound to the biotinylated target DNA (A) or a non-specific biotinylated control DNA (B)

Courtesy of D. Mulholland, Vancouver, Canada

Isolation of a transcription factor

Coomassie Blue-stained PAGE gel

E.coli extract was incubated with a biotinylated operator followed by trapping the protein-DNA complexes to streptavidin

Lane 1: flow-through

Lane 2: eluate

from E. coli expressing the recombinant factor

M 1 2

30 kDtranscription factor

Translation control studies with µMACSTM Streptavidin Kit

The probe for isolation of RNA binding proteins was generated by a full length transcript of the RNA, hybridized to a specific 3' biotinylated ss-oligonucleotid bound to µMACS Streptavidin MicroBeads.

Purification of RNA binding proteins

RNA / oligo / µMACS MicroBead complex

Complimentary 3' biotinylated oligo

RNA probe

MACS MicroBead

Translation control studies with µMACSTM Streptavidin Kit

Silver Staining of SDS-PAGE. Yeast crude extract was precleared with heparin agarose and subsequently incubated with the RNA/mutant RNA-µMACS Streptavidin MicroBead probe.

Purification of RNA binding proteins

Full length RNA column Mutant RNA column

µMACS in proteomics research

• Protein isolation

• Interaction partner isolation

• Protein complex isolation

• Enzymatic, on-column reactions with the temperature-controlled thermoMACS Separator