Welcome toTechniques in Molecular BiologyVTMC831May 2006

May 1Introduction to the classBackground on class exercises - The Luman and Zgangfei storyExercises for week 1Lecture - Detection of ProteinsLaboratory exercise for the day

The Luman - Zhangfei StoryLumanZhangfei

Oct 1VP16HCFIm EarlyEarlyLateLATTAATGARAT

HCF-binding proteinsD/EHxYLumanZhangfeiE2F4Krox 20HPIP(Brn3a)Set1NCHCFVP16SP1GABPMiz1Sin3-HDACchromatinprot. phosPDCD2PGC1

Luman

intracellular location of LumanLuman in cultured rat neuron

Luman may sequester HCF in the cytoplasm of neuronsH and ELumanHCF

Zhangfei

Zhangfei is expressed in neuronsanti-ZFZF absorbed anti-ZFcerebrumsensory ganglion

PC12 cells - an in vitro model for studying neuronsNerve growth factor

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

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)

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

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

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

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).

SDS-PAGE

PGC1 - nuclear hormone co-activatorHPIP - nuclear export factorSet1 - chromatin remodeling complexSin3 - Histone deacetylasePDCD2 - programmed cell death regulator