LOCALIZATION OF A MOLECULE - placing in space and or time. -Answering where and/when.

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LOCALIZATION OF A MOLECULE- placing in space and
or time.-Answering where and/when.
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Localize with respect to WHAT?

• TIME - embryogenesis, cell cycle, post
  stimulation with calcium, enzymes, etc.
• SPACE - within a cell, within a tissue, within an
  organism, within a genome

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Methods for localization

• PROTEIN
• Immunolocalization
• endogenous or tagged?
• Direct detection via gfp or enzyme (b-gal)
• Gold Particle
• Cell fractionation
• RNA
• In Situ Hybridization in cells, tissues,
  embryos
• DNA
• FISH

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IMMUNOLOCALIZATION

• ANTIBODIES
• what are they?
• what do they look like?
• where in the antibody does the specificity come
  from?
• - EPITOPES
• what are they?
• what do they look like?

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Variable
Variable
Constant
Light chain
Heavy chain
Diversity comes from DNA recombination in heavy
and light chain loci.
Heavy chain constant region determines isotype of
antibody IgG, IgM, IgA, IgE.
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EPITOPESA few amino acid - 8ExamplesFLAG
Asp-Tyr-Lys-Asp-Asp-Asp-Asp-LysMyc
Glu-Glu-Lys-Ile-Ser-Glu-Glu-Asp-Leu-AsnV5,
His,GST
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HOW TO MAKE AN ANTIBODY 101 (listen qual exams!)

• What to start with?
• How pure is it?
• Then what.

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POLYCLONAL VS MONOCLONAL
Whats the difference?
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Sacrifice animal, Collect b cells from
spleen Fuse with myeloma cells to produce
hybridomas (immortalized cell lines) Screen lots
of hybridomas for ones that react to antigen of
choice.
Collect blood
Use sera or purify more (aff. Chrom)
Advantages vs Disadvantages
Is this a single ab?
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• ADVANTAGE MONOCLONAL
• specificity!!
• Generate large amounts
• Can immunize with complex mix of immunogen and
  screen for clone of choice
• DISADVANTAGE MONOCLONAL
• -time
• -expense!
• -ONLY ONE species of antibody in the mix..

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How to characterize a new antibody.

• TITER AND SPECIFICITY!!!
• How to test each of these?

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IS IT CORRECT?

• ARTIFACTS?
• CONTROLS?

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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

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5
3
sp6
YFG

1. Make Antisense RNA labeled
2. Make Sense RNA labeled (dig, fluoro, s35,etc)

t7
Hybridize to sample
Detect if radioactive
If non rad detect using antibody against RNA tag
that is conjugated to an enzyme (AP, HRP)
Color reaction
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Rt-pcr
Harvest Tissue
DNAase, Phenol (i.e. get rid of dna and proteins
Make cDNA with reverse transcriptase (RT)
Radioactive or Nonradioactive PCR with YF primers
Also Rnase Protection..
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LOCALIZATION OF DNA

• FISH
• Radiation hybrid or genetic linkage mapping.

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Fluoresence In Situ Hyb (FISH)
Label probe DNA with fluorescent dye
Denature and Hybridize to sample (cells)
Localize by microscopy to chromosomes
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Cell fractionation

• Fractionation followed by assay of fractions for
  molecules of interest.
• western could be used to detect protein.
• Northern or rtpcr could be used to detect RNA.
• - Need markers for cell components to assess
  purity.