Transcript 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
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