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

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Presentation transcript:

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?

Light chain Heavy chain Variable Constant 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-Asp- Asp-Lys Myc: Glu-Glu-Lys-Ile-Ser-Glu-Glu- Asp-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?

Collect blood Use sera or purify more (aff. Chrom) 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. Advantages vs Disadvantages Is this a single ab?

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

sp6 t7 5’ 3’ YFG1.Make Antisense RNA labeled 2.Make Sense RNA labeled (dig, fluoro, s35,etc) Hybridize to sample If non rad detect using antibody against RNA tag that is conjugated to an enzyme (AP, HRP) Color reaction Detect if radioactive

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.