Transcript Bio4751signaltransductionTechniques

How can we detect measure gene/protein activation?
DNA
RNA
Southern
Polymerase Chain reaction (PCR)
Note- These detects gene, not expression
Northern
RT-PCR
Primer extension
Protein
Microarray
RNase Protection
Western (immunoblot)
How do we examine DNA-protein interactions?
Mad
Electrophoretic Mobility Shift Assay (EMSA) (aka gel shift)
DNaseI protection
Photo-crosslinking
How do we examine protein-protein interactions?
GST pull-down
EMSA Supershift
Co-immunoprecipitation
Techniques to know to understand signal
transduction
1. Southern- Detect DNA only
2. Northern- Detect RNA
3. Microarray- Detect RNA of 100s of expressed
genes
4. RT-PCR ( Reverse transcription polymerase
chain reaction- to detect RNA)
5. Western (Immunoblot)- Detect protein
6. Immunostaining- Detect proteins in situ
7. EMSA- protein-DNA interactions
8. Supershift – Protein-protein interactions
9. Co-immunopreciptiation- Protein-protein
interactions
Separation of DNA/RNA/Proteins by gel electrophoresis
(-) Electrode Protein mixture
(+)
Electrode
Polyacrylamide Gel
Direction of current
Agarose gel electrophoresis used for RNA and DNA separation
Polyacrylamide gel electrophoresis is used for protein separation
Large proteins
Direction
of
current
Small proteins
RNA detection
1. Southern analysis- to determine if gene is present/defective
2. Northern transfer and analysis
1
2
3
1
1. Northern
transfer to nylon
membrane
28S
18S
2
3
Ras detected
2. Hybridize with
Ras DNA probe
3. Wash and
expose to film
Stained gel
Film exposed to
probed membrane
3. Microarrays- monitor expression of several genes at once
Genome-wide analysis- yeast
RNA detection
4. RT-Polymerase
Chain Reaction
(RT-PCR)
Fig. 4.17
One copy mRNA
many copies DNA
4. RT-Polymerase Chain Reaction (RT-PCR)
Example- detect RasGAP gene RNA isolated for cells
Cell mRNA
cDNA
Load onto agarose gel
Agarose gel
electrophoresis
RT-PCR
Components of a Western analysis
Protein detection
5. Western Analysis
Polyacrylamide gel
elecrophoresis,
transfer to nylon
membrane
1. Protein separation
2. Detection
Secondary Ab
(Anti-rabbit Ab)
Alkaline
peroxidase
Primary Ab
(Rabbit Anti-Ras)
RAS
Ras protein
If use antibody to phospho-Ras,
then detect slower migrating
phopho-Ras
5. Western Analysis- Example
After
5000
1000
500
100
Imidazole (mM)
Wash
0
25
Lysate
Nickel Column,
Imidizole elution
Flowthrough
Before
Bacterial Cell Lysate
pTAT-HNF4
132 kD
86 kD
43 kD
32 kD
17 kD
Anti-HA
Anti-HNF4
Probe for specific protein
Fraction
Liver
Onto nylon membrane
PAGE Ion Exchange
using antibodies
2
Dialysis
132 kD
3
4
5. Western Analysis
How do we detect activated (phosphorylated) proteins?
If use anti-phosphotyrosine (anti-pY) antibody…
….observe several phosphorylated proteins
If use anti-phosphoERK (anti-p-ERK) antibody…
….observe only phosphorylated ERK
If use anti-ERK (anti-p-ERK) antibody…
….observe total ERK
6. Western
Immunostaining
Analysis
Detect protein inside of a cell?
Enzyme or fluorescent
molecule
Secondary Ab
(Anti-rabbit Ab)
Primary Ab
(Rabbit Anti-Ras)
RAS
Cell
6. Immunostaining- example 1
GP73 in human tissues
Colon
Lung
Kidney
Prostate
GCH Liver
Liver
Immunohistochemistry
Kladney et. al. Gene 249:53 2000
6. Immunostaining- example 2
Upregulation of GP73 expression by
adenovirus infection is specific
7. EMSA – Detect
Detects
DNA
DNA–protein
–protein complex
interaction
Cell lysate + Radiolabeled DNA
DNA-protein complex
Commonly used to
identify transcription
factor (SMAD, STAT,
NF-kB, etc.) binding
to promoter DNA
Unbound DNA
NF-kB
8. Supershift – “An EMSA with antibody added”
Cell lysate + Radiolabeled DNA
+ antibody to protein of interest
Anti-NF-kB
LPS
U C LPS CHx +CHx
- - - + - +-
supershift
NF-kB
Used to
1. Verifies identity
of protein in
DNA/protein
complex
2.
Identify other
proteins in the
DNA-protein
complex
NF-kB
+
9. Co-immunopreciptiation- Protein-protein interactions
receptor
Grb2
Ras
Cell
RasGEF
Do these interact??
Lyse cell
Primary Ab
Immunoprecipitate
(Rabbit Anti-RasGEF) with anti-RasGEF Ab
RasGEF
PAGE
Transfer to membrane
Grb2 protein
Probe with anti- Grb2
antibody and
secondary antibody
Only detect Grb 2 only if it is
binding to RasGEF in cells