Welcome toTechniques in Molecular

BiologyVTMC831

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

Oct 1

VP16

HCF

Im Early

Early

Late

LAT

TAATGARAT

HCF-binding proteins

D/EHxY

Luman

Zhangfei

E2F4

Krox 20

HPIP

(Brn3a)

Set1

N C

HCF

VP16SP1

GABPMiz1

Sin3-HDACchromatin

prot. phos

PDCD2PGC1

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 ZF absorbed anti-ZF

cerebrum

sensory ganglion

Nerve growth factor

PC12 cells - an in vitro model for studying neurons

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 S70-S74, 2005

Detection of proteins in cells

• Giepmans 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-PAGE

• transfer to a membrane

• detection by antibody (fluorescence or enzyme-linked chemiluminescence or colour).

SDS-PAGE