LOCALIZING MOLECULES. 1. WHAT MOLECULES? LOCALIZING MOLECULES 1. WHAT MOLECULES? 2. WITH RESPECT TO WHAT?

LOCALIZING MOLECULES

LOCALIZING MOLECULES

1. WHAT MOLECULES?

LOCALIZING MOLECULES

1. WHAT MOLECULES?2. WITH RESPECT TO WHAT?

LOCALIZING MOLECULES

1. WHAT MOLECULES?2. WITH RESPECT TO WHAT? SPACE: Relative to other molecules,

the genome, organelles, cells, tissues, whole organisms.

LOCALIZING MOLECULES

1. WHAT MOLECULES?2. WITH RESPECT TO WHAT? SPACE: Relative to other molecules,

the genome, organelles, cells, tissues, whole organisms.

TIME: Cell cycle, cell differentiation, embryogenesis, post-stimulation with calcium,…

LOCALIZING MOLECULES

1. WHAT MOLECULES?2. WITH RESPECT TO WHAT?3. WHY BOTHER?

LOCALIZING MOLECULES

1. WHAT MOLECULES?2. WITH RESPECT TO WHAT?3. WHY BOTHER? Knowing where and when allows

inferences as to interactions and functions.

Methods for localization

• PROTEIN Direct detection via GFP (etc.) or enzyme

(-gal, GUS, etc.) fusion Immunolocalization (endogenous or

tagged protein) Cell fractionation

• RNA In situ hybridization in cells, tissues,

embryos• DNA

FISH

Swam for Harvard; won 2008 Chemistry Nobel Prize for this work.

Now works for Mike Snyder at the Stanford Genomics Center; expressed fluorescent GFP in E. coli as her ROTATION PROJECT.

Cloned and sequenced the Aequorea victoria GFP gene based on Shimomura’s earlier painstaking purification of the protein from jellyfish extracts. Now works for an auto dealership in Huntsville, AL.

Science 263: 802-805 (1994)

Direct Localization

GFP (green fluorescent protein: 30 kDa)

Roger Tsien, UCSD (CFP, YFP, RFP, tomato,…)

IMMUNOLOCALIZATION

ANTIBODIES:

EPITOPES:

Light chainHeavy chain

Constant

Diversity comes from DNA recombination in heavy and light chain loci

Heavy chain constant region determines isotype of antibody… IgG, IgM, IgA, IgE.

Antibodies

VariableVariable

EPITOPES

Examples of epitope tags:

FLAG: Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys

Myc: Glu-Glu-Lys-Ile-Ser-Glu-Glu-Asp-Leu-Asn

HA, V5, EE, His6 (GST, GFP)

1. A few amino acids (~6-12)2. A bit of sugar (glycosyl groups)3. Some other chemical shape that can

be recognized (DNA with BrdU in it)

HOW TO MAKE AN ANTIBODY (listen… qualifying exams!)

What to start with?

How pure is it?

HOW TO MAKE AN ANTIBODY (listen… qualifying exams!)

What to start with?

How pure is it?

1. Purified protein (or polysaccharide, etc.)

2. Synthetic peptide (conjugated to something big)

3. Purified fusion protein (typically from E. coli)

POLYCLONAL VS. MONOCLONAL

What’s the difference?

Collect blood

Use serum or purify more?

(affinity methods)

Is this a single Ab?

Polyclonal

Sacrifice animal

Collect B cells from spleen

Fuse with myeloma cells to produce hybridomas (immortalized cell lines)

Screen lots of hybridoma clones for ones that react to antigen of choice

Monoclonal

ADVANTAGES OF MONOCLONAL:1. Specificity!!2. Can generate large amounts3. Can immunize with a complex mix of

immunogens and screen for clone(s) of choice

DISADVANTAGES OF MONOCLONAL:4. Time5. Expense!6. ONLY ONE species of antibody in the mix

(But do you know its epitope?)

ADVANTAGES OF POLYCLONAL:1. Easy2. Relatively rapid to generate3. High potency for both IF and IP

(several types of antibodies)

DISADVANTAGES OF POLYCLONAL:4. Possibly lower amounts and lack of supply

in perpetuity (but the animal matters)5. Lower specificity6. Do not know the exact epitope (unless you

started with a synthetic peptide)

IMMUNOSTAININGDIRECT VS. INDIRECT Signal-strength issues

ARTIFACTS? Cross-reactions

CONTROLS? Pre-immune vs. immune serum

Affinity purification!!

IMMUNOLOCALIZATIONVS.

GFP(etc.)-TAGGING

IMMUNOLOCALIZATIONVS.

GFP(etc.)-TAGGING

1. Fixed vs. living cells2. Potential for real-time (time-lapse)

analysis3. Potential for cross-reaction artifacts4. Potential for IPs and Western blots5. Potential for analysis at EM level6. Signal-strength issues7. Organisms to which can be applied

CELL FRACTIONATION

Fractionation followed by assay of fractions for molecules of interest

Western-blot to detect protein

Need markers for cell components to assess purity (PM, nucleus, ER, Golgi, cytoplasm, mitochondria…)

Cell Fractionation

Fractionation

(nuclear marker)

(cytoplasmic marker)

Localization of RNA

In situ hybridization(cells, tissues, whole embryos…)

What are the appropriate controls? What part of a protein coding gene

would make the best probe?

Northern Blot or RTPCR or RNAse protection on RNA from different sources/tissues

sp6t7

5’ 3’YFG 1. Make Antisense RNA labeled

2. Make Sense RNA labeled (digoxigenin, fluoro, S35…)

Hybridize to sample

Detect using antibody against RNA tag that is conjugated to an enzyme (AP, HRP)

Color reaction

Detect if radioactive

In situ hybridization

E10.5 wt E11.5 PERP-null

In situ hybridization with a digoxigenin-labeled antisense probe

Laura Attardi’s lab

LOCALIZATION OF DNAFluorescence In Situ Hyb (FISH)

Label probe DNA with fluorescent dye

Localize by microscopy to chromosomes

Denature and Hybridize to

sample (cells)