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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 HCF Early VP16 Oct 1 TAATGARAT Im Early Late LAT HCF-binding proteins HCF N C SP1 VP16 D/EHxY Miz1 PGC1 Luman Krox 20 Zhangfei Set1 HPIP E2F4 (Brn3a) Sin3-HDAC PDCD2 GABP prot. phos chromatin Luman intracellular location of Luman Luman Calnexin Luman in cultured rat neuron Luman may sequester HCF in the cytoplasm of neurons H and E Luman HCF Zhangfei Zhangfei is expressed in neurons anti-ZF cerebrum sensory ganglion ZF absorbed anti-ZF PC12 cells - an in vitro model for studying neurons Nerve growth factor Exercise 1 Is Zhangfei (protein and RNA) differentially expressed in undifferentiated and differentiated PC12 cells? 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) Antibodies from eggs • Lucyna Cova. DNA-designed avian IgY antibodies: novel tools for research, diagnostics and therapy. Journal of Clinical Virology. 34, suppl 1 S70S74, 2005 Detection of proteins in cells • Giepmans et al. The fluorescent toolbox for assessing protein location and function. Science 312, April 14, 2006. Fig. 3. Parallel application of targeting methods and fluorophores B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Published by AAAS 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). SDS-PAGE