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. __________- Detect DNA only
2. ___________- Detect RNA
3. ___________- Detect RNA of ____ of
expressed genes
4. ________ ( Reverse transcription polymerase
chain reaction- to detect RNA)
5. ________________________ Detect protein
6. _______________- Detect proteins in situ
7. _________- protein-DNA interactions
8. ________– Protein-protein interactions
9. ______________- Protein-protein interactions
Separation of DNA/RNA/Proteins by gel electrophoresis
(-) Electrode Protein mixture
(+)
Electrode
Polyacrylamide Gel
Direction of current
_________________________ used for RNA and DNA separation
________________________ 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 _____
membrane
28S
18S
2
3
Ras detected
2. Hybridize with
Ras _____ probe
3. Wash and
expose to _____
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
_____
Load onto __________ 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
132 kD
86 kD
43 kD
32 kD
17 kD
5000
1000
500
100
Imidazole (mM)
Wash
0
25
Lysate
Nickel Column,
Imidizole elution
Flowthrough
Before
Bacterial Cell Lysate
pTAT-HNF4
Anti-HA
Anti-HNF4
Probe for specific protein
using antibodies
Fraction
2
Dialysis
132 kD
3
4
Liver
Onto nylon membrane
PAGE Ion Exchange
5. Western Analysis
How do we detect activated (phosphorylated) proteins?
If use anti-phosphotyrosine (anti-pY) antibody…
….observe ______________________ proteins
If use anti-phosphoERK (anti-p-ERK) antibody…
….observe only ____________________
If use anti-ERK (anti-p-ERK) antibody…
….observe ___________________
6. Western
Immunostaining
Analysis
Detect protein inside of a cell?
Enzyme or __________
molecule
__________ Ab
(Anti-rabbit Ab)
__________ 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 + ____________ DNA
DNA-protein complex
Commonly used to
identify ___________
factor (SMAD, STAT,
NF-kB, etc.) binding
to ________________
Unbound DNA
NF-kB
8. Supershift – “An EMSA with antibody added”
Cell lysate + Radiolabeled DNA
+ _________ to protein of interest
Anti-NF-kB
LPS
U C LPS CHx +CHx
- - - + - +-
supershift
NF-kB
Used to
1. Verifies _______
of protein in
DNA/protein
complex
2. Identify ______
________ 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