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LOCALIZING MOLECULES

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?

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LOCALIZING MOLECULES

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  1. LOCALIZING MOLECULES

  2. LOCALIZING MOLECULES 1. WHAT MOLECULES?

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

  4. LOCALIZING MOLECULES 1. WHAT MOLECULES? 2. WITH RESPECT TO WHAT? SPACE: Relative to other molecules, the genome, organelles, cells, tissues, whole organisms.

  5. 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,…

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

  7. LOCALIZING MOLECULES 1. WHAT MOLECULES? 2. WITH RESPECT TO WHAT? 3. WHY BOTHER? Knowing where and when allows inferences as to interactions and functions.

  8. 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

  9. Science263: 802-805 (1994) 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.

  10. Direct Localization GFP (green fluorescent protein: 30 kDa) Roger Tsien, UCSD (CFP, YFP, RFP, tomato,…)

  11. IMMUNOLOCALIZATION ANTIBODIES: EPITOPES:

  12. Antibodies 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.

  13. EPITOPES • A few amino acids (~6-12) • A bit of sugar (glycosyl groups) • Some other chemical shape that can be recognized (DNA with BrdU in it) 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)

  14. HOW TO MAKE AN ANTIBODY (listen… qualifying exams!) What to start with? How pure is it?

  15. HOW TO MAKE AN ANTIBODY (listen… qualifying exams!) What to start with? How pure is it? • Purified protein (or polysaccharide, etc.) • Synthetic peptide (conjugated to something big) • Purified fusion protein (typically from E. coli)

  16. POLYCLONAL VS. MONOCLONAL What’s the difference?

  17. Polyclonal Monoclonal Sacrifice animal Collect B cells from spleen Fuse with myeloma cells to produce hybridomas (immortalized cell lines) Screen lots of hybridomaclones for ones that react to antigen of choice Collect blood Use serum or purify more? (affinity methods) Is this a single Ab?

  18. ADVANTAGES OF MONOCLONAL: • Specificity!! • Can generate large amounts • Can immunize with a complex mix of immunogensand screen for clone(s) of choice DISADVANTAGES OF MONOCLONAL: • Time • Expense! • ONLY ONE species of antibody in the mix (But do you know its epitope?)

  19. ADVANTAGES OF POLYCLONAL: • Easy • Relatively rapid to generate • High potency for both IF and IP (several types of antibodies) DISADVANTAGES OF POLYCLONAL: • Possibly lower amounts and lack of supply in perpetuity (but the animal matters) • Lower specificity • Do not know the exact epitope (unless you started with a synthetic peptide)

  20. IMMUNOSTAINING DIRECT VS. INDIRECT Signal-strength issues ARTIFACTS? Cross-reactions CONTROLS? Pre-immune vs. immune serum Affinity purification!!

  21. IMMUNOLOCALIZATIONVS.GFP(etc.)-TAGGING

  22. IMMUNOLOCALIZATIONVS.GFP(etc.)-TAGGING • 1. Fixed vs. living cells • Potential for real-time (time-lapse) analysis • Potential for cross-reaction artifacts • Potential for IPs and Western blots • Potential for analysis at EM level • Signal-strength issues • Organisms to which can be applied

  23. 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…)

  24. Cell Fractionation

  25. Fractionation (cytoplasmic marker) (nuclear marker)

  26. 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

  27. In situ hybridization 5’ 3’ YFG sp6 Make Antisense RNA labeled Make Sense RNA labeled (digoxigenin, fluoro, S35…) t7 Hybridize to sample Detect if radioactive Detect using antibody against RNA tag that is conjugated to an enzyme (AP, HRP) Color reaction

  28. In situ hybridization with a digoxigenin-labeled antisense probe E10.5 wt E11.5 PERP-null Laura Attardi’s lab

  29. LOCALIZATION OF DNAFluorescence 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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