Download LOCALIZATION OF A MOLECULE

LOCALIZATION OF A
MOLECULE
- placing in space and or time.
-Answering where and/when.
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
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
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?
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.
EPITOPES
A few amino acid - 8
Examples:
FLAG: Asp-Tyr-Lys-Asp-Asp-AspAsp-Lys
Myc: Glu-Glu-Lys-Ile-Ser-Glu-GluAsp-Leu-Asn
V5, His,GST
HOW TO MAKE AN
ANTIBODY 101
(listen… qual exams!)
• What to start with?
• How pure is it?
• Then what….
POLYCLONAL VS
MONOCLONAL
Whats the difference?
Sacrifice animal,
Collect b cells from spleen
Collect blood
Fuse with myeloma cells to
produce hybridomas
(immortalized cell lines)
Use sera or
purify more
(aff. Chrom)
Screen lots of hybridomas for
ones that react to antigen of
choice.
Is this a single ab?
Advantages vs Disadvantages
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..
How to characterize a new
antibody….
TITER AND SPECIFICITY!!!
How to test each of these?
IS IT CORRECT?
• ARTIFACTS?
• CONTROLS?
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
5’
t7
3’
YFG
sp6
1. Make Antisense RNA labeled
2. Make Sense RNA labeled (dig,
fluoro, s35,etc)
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
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…..
LOCALIZATION OF DNA
• FISH
• Radiation hybrid or genetic linkage
mapping.
Fluoresence In Situ Hyb (FISH)
Label probe DNA with
fluorescent dye
Denature and
Hybridize to sample
(cells)
Localize by
microscopy to
chromosomes
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.