how can we detect measure gene/protein activation?
DESCRIPTION
Mad. How can we detect measure gene/protein activation?. DNA. Southern. Polymerase Chain reaction (PCR). Note- These detects gene, not expression. Microarray. RT-PCR. RNA. Northern. Primer extension. RNase Protection. Protein. Western (immunoblot). - PowerPoint PPT PresentationTRANSCRIPT
How can we detect measure gene/protein activation?
DNA
RNA
Protein
Northern
Western (immunoblot)
RNase Protection
How do we examine DNA-protein interactions?
Mad
Electrophoretic Mobility Shift Assay (EMSA) (aka gel shift)
DNaseI protection
How do we examine protein-protein interactions?
GST pull-down
EMSA Supershift
Co-immunoprecipitation
Primer extension
Photo-crosslinking
Southern
Note- These detects gene, not expression
RT-PCR
Polymerase Chain reaction (PCR)
Microarray
Techniques to know to understand signal transduction
1. __________- Detect DNA only2. ___________- Detect RNA3. ___________- Detect RNA of ____ of expressed genes4. ________ ( Reverse transcription polymerase chain
reaction- to detect RNA)5. ________________________ Detect protein6. _______________- Detect proteins in situ7. _________- protein-DNA interactions8. ________– Protein-protein interactions9. ______________- Protein-protein interactions
Polyacrylamide Gel
Protein mixture (+) Electrode
(-) Electrode
Direction of current
Direction of
current Small proteins
Large proteins
Separation of DNA/RNA/Proteins by gel electrophoresis
_________________________ used for RNA and DNA separation
________________________ gel electrophoresis is used for protein separation
28S
18S
1 2 3 1 2 3
Stained gel Film exposed to probed membrane
1. Northern transfer to _____
membrane
2. Hybridize with Ras _____ probe
3. Wash and expose to _____
2. Northern transfer and analysis
Ras detected
RNA detection
1. Southern analysis- to determine if gene is present/defective
Genome-wide analysis- yeast
3. Microarrays- monitor expression of several genes at once
One copy mRNA
many copies DNAFig. 4.17
RNA detection4. RT-Polymerase
Chain Reaction(RT-PCR)
RT-PCR
4. RT-Polymerase Chain Reaction (RT-PCR)
Example- detect RasGAP gene RNA isolated for cells
Agarose gel electrophoresis
Cell mRNA _____ Load onto __________ gel
Primary Ab (Rabbit Anti-Ras)
RAS
Secondary Ab(Anti-rabbit Ab)
Alkaline peroxidase
Components of a Western analysis
Polyacrylamide gel elecrophoresis, transfer to nylon
membrane
1. Protein separation
2. Detection
Ras protein
If use antibody to phospho-Ras, then detect slower migrating phopho-Ras
Protein detection5. Western Analysis
Anti-HNF4
Anti-HA
Lys
ate
Flow
thro
ugh
Wa s
h
500
1000
5000
Imidazole (mM)
Anti-HA
Liv
er
Fraction
2 3 4
Anti-HNF4
Bacterial Cell LysatepTAT-HNF4
Nickel Column,Imidizole elution
Ion Exchange
Dialysis
PD10 column
43 kD86 kD
132 kD
17 kD
32 kD
43 kD86 kD
132 kD
17 kD
32 kD
55 kD
90 kD132 kD
43 kD
Anti-HNF4
Anti-HA
Lys
ate
Flow
thro
ugh
Wa s
h
500
1000
5000
Imidazole (mM)
Anti-HA
Liv
er
Fraction
2 3 4
Anti-HNF4
Bacterial Cell LysatepTAT-HNF4
Nickel Column,Imidizole elution
Ion Exchange
Dialysis
PD10 column
43 kD86 kD
132 kD
17 kD
32 kD
43 kD86 kD
132 kD
17 kD
32 kD
43 kD86 kD
132 kD
17 kD
32 kD
43 kD86 kD
132 kD
17 kD
32 kD
55 kD
90 kD132 kD
43 kD
Before After
PAGE Onto nylon membrane Probe for specific protein using antibodies
5. Western Analysis- Example
If use anti-phosphotyrosine (anti-pY) antibody…
….observe ______________________ proteins
If use anti-phosphoERK (anti-p-ERK) antibody…
….observe only ____________________
How do we detect activated (phosphorylated) proteins?
If use anti-ERK (anti-p-ERK) antibody…
….observe ___________________
5. Western Analysis
6. Western Analysis
Detect protein inside of a cell?
__________ Ab (Rabbit Anti-Ras)
RAS
__________ Ab(Anti-rabbit Ab)
Enzyme or __________ molecule
Cell
6. Immunostaining
GP73 in human tissues
Colon
Kidney
Liver
Lung
Prostate
GCH Liver
ImmunohistochemistryKladney et. al. Gene 249:53 2000
6. Immunostaining- example 1
Upregulation of GP73 expression byadenovirus infection is specific
6. Immunostaining- example 2
NF-B
– Ø
– LPS
– CH
x–
LPS+C
Hx
– TN
FUnbound DNA
DNA-protein complex
7. EMSA – Detect DNA –protein complex
Cell lysate + ____________ DNA
7. EMSA – Detects DNA–protein interaction
Commonly used to identify ___________ factor (SMAD, STAT, NF-kB, etc.) binding to ________________
Anti-NF-kB
NF-B
supershift
NF-B
--LPS CHx
LPS+CHx
+ + +--C
-U
8. Supershift – “An EMSA with antibody added”
Cell lysate + Radiolabeled DNA
+ _________ to protein of interest
1. Verifies _______ of protein in DNA/protein complex
Used to
2. Identify ______ ________ in the DNA-protein complex
RasGEF
Grb2 Ras
Immunoprecipitate with anti-RasGEF Ab
PAGE
Probe with anti- Grb2 antibody and
secondary antibody
Transfer to membrane
9. Co-immunopreciptiation- Protein-protein interactions
receptor
Primary Ab (Rabbit Anti-RasGEF)
RasGEF
Do these interact??Lyse cell
Grb2 protein
Only detect Grb 2 only if it is binding to RasGEF in cells
Cell