Download - Welcome to Techniques in Molecular Biology
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Welcome toTechniques in Molecular BiologyVTMC831May 2006
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May 1Introduction to the classBackground on class exercises - The Luman and Zgangfei storyExercises for week 1Lecture - Detection of ProteinsLaboratory exercise for the day
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The Luman - Zhangfei StoryLumanZhangfei
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Oct 1VP16HCFIm EarlyEarlyLateLATTAATGARAT
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HCF-binding proteinsD/EHxYLumanZhangfeiE2F4Krox 20HPIP(Brn3a)Set1NCHCFVP16SP1GABPMiz1Sin3-HDACchromatinprot. phosPDCD2PGC1
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Luman
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intracellular location of LumanLuman in cultured rat neuron
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Luman may sequester HCF in the cytoplasm of neuronsH and ELumanHCF
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Zhangfei
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Zhangfei is expressed in neuronsanti-ZFZF absorbed anti-ZFcerebrumsensory ganglion
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PC12 cells - an in vitro model for studying neuronsNerve growth factor
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Exercise 1Is Zhangfei (protein and RNA) differentially expressed in undifferentiated and differentiated PC12 cells?
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Detection of Proteinsantibody probes (detected by secondary antibody linked to an enzyme or a fluorescent tag)immunization with whole proteinpurification of proteinfrom tissues or cultured cellsrecombinant proteinDNA immunization of birds - IgYepitope tagsgenetically attachedfluorescent proteinsfluor-binding tags (tetracysteine-biarsenical system)
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Antibodies from eggsLucyna Cova. DNA-designed avian IgY antibodies: novel tools for research, diagnostics and therapy. Journal of Clinical Virology. 34, suppl 1 S70-S74, 2005
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Detection of proteins in cellsGiepmans et al. The fluorescent toolbox for assessing protein location and function. Science 312, April 14, 2006.
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Published by AAAS B. N. G. Giepmans et al., Science 312, 217 -224 (2006) Fig. 3. Parallel application of targeting methods and fluorophores
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Detection of proteins in mixtures (cell/tissue lysates)separation of proteins by SDS-PAGEtransfer to a membrane detection by antibody (fluorescence or enzyme-linked chemiluminescence or colour).
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SDS-PAGE
PGC1 - nuclear hormone co-activatorHPIP - nuclear export factorSet1 - chromatin remodeling complexSin3 - Histone deacetylasePDCD2 - programmed cell death regulator