Post on 24-Feb-2016
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LOCALIZING MOLECULES
LOCALIZING MOLECULES1. WHAT MOLECULES?
LOCALIZING MOLECULES1. WHAT MOLECULES?2. WITH RESPECT TO WHAT?
LOCALIZING MOLECULES1. WHAT MOLECULES?2. WITH RESPECT TO WHAT? SPACE: Relative to other molecules,
the genome, organelles, cells, tissues, whole organisms.
LOCALIZING MOLECULES1. 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 MOLECULES1. WHAT MOLECULES?2. WITH RESPECT TO WHAT?3. WHY BOTHER?
LOCALIZING MOLECULES1. 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 LocalizationGFP (green fluorescent protein: 30 kDa)
Roger Tsien, UCSD (CFP, YFP, RFP, tomato,…)
IMMUNOLOCALIZATIONANTIBODIES:
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.
AntibodiesVariableVariable
EPITOPES
Examples of epitope tags:
FLAG: Asp-Tyr-Lys-Asp-Asp-Asp-Asp-LysMyc: Glu-Glu-Lys-Ile-Ser-Glu-Glu-Asp-Leu-AsnHA, 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 animalCollect B cells from spleenFuse 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 issuesARTIFACTS? Cross-reactionsCONTROLS? Pre-immune vs. immune serum Affinity purification!!
IMMUNOLOCALIZATIONVS.
GFP(etc.)-TAGGING
IMMUNOLOCALIZATIONVS.
GFP(etc.)-TAGGING1. 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 RNAIn 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)