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Welcome to Techniques in Molecular Biology

Welcome to Techniques in Molecular Biology. VTMC831 May 2006. May 1. Introduction to the class Background on class exercises - The Luman and Zgangfei story Exercises for week 1 Lecture - Detection of Proteins Laboratory exercise for the day. The Luman - Zhangfei Story. Luman. Zhangfei.

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Welcome to Techniques in Molecular Biology

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  1. Welcome toTechniques in Molecular Biology VTMC831 May 2006

  2. May 1 • Introduction to the class • Background on class exercises - The Luman and Zgangfei story • Exercises for week 1 • Lecture - Detection of Proteins • Laboratory exercise for the day

  3. The Luman - Zhangfei Story Luman Zhangfei

  4. HCF Early VP16 Oct 1 Im Early TAATGARAT Late LAT

  5. HCF-binding proteins HCF N C SP1 Sin3-HDAC D/EHxY VP16 chromatin PDCD2 Miz1 GABP PGC1 Luman prot. phos Krox 20 Zhangfei HPIP Set1 E2F4 (Brn3a)

  6. Luman

  7. Luman Calnexin intracellular location of Luman Luman in cultured rat neuron

  8. Luman may sequester HCF in the cytoplasm of neurons H and E Luman HCF

  9. Zhangfei

  10. Zhangfei is expressed in neurons anti-ZF ZF absorbed anti-ZF cerebrum sensory ganglion

  11. PC12 cells - an in vitro model for studying neurons Nerve growth factor

  12. Exercise 1 Is Zhangfei (protein and RNA) differentially expressed in undifferentiated and differentiated PC12 cells?

  13. Detection of Proteins • antibody probes (detected by secondary antibody linked to an enzyme or a fluorescent tag) • immunization with whole protein • purification of protein • from tissues or cultured cells • recombinant protein • DNA immunization of birds - IgY • epitope tags • genetically attached • fluorescent proteins • fluor-binding tags (tetracysteine-biarsenical system)

  14. Antibodies from eggs • Lucyna Cova. DNA-designed avian IgY antibodies: novel tools for research, diagnostics and therapy. Journal of Clinical Virology. 34, suppl 1 S70-S74, 2005

  15. Detection of proteins in cells • Giepmans et al. The fluorescent toolbox for assessing protein location and function. Science 312, April 14, 2006.

  16. Fig. 3. Parallel application of targeting methods and fluorophores B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Published by AAAS

  17. Detection of proteins in mixtures (cell/tissue lysates) • separation of proteins by SDS-PAGE • transfer to a membrane • detection by antibody (fluorescence or enzyme-linked chemiluminescence or colour).

  18. SDS-PAGE

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