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