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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 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 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