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 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

Polyacrylamide Gel Protein mixture (+) Electrode (-) Electrode 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 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 RNA detection 4. 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 detection 5. Western Analysis

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 Immunohistochemistry Kladney et. al. Gene 249: Immunostaining- example 1

Upregulation of GP73 expression by adenovirus infection is specific 6. Immunostaining- example 2

NF-  B – Ø – LPS – CHx – LPS+CHx – TNF Unbound 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