How can we detect measure gene/protein activation?

1. 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) How do we examine DNA-protein interactions? Electrophoretic Mobility Shift Assay (EMSA) (aka gel shift) DNaseI protection Photo-crosslinking How do we examine protein-protein interactions? GST pull-down Co-immunoprecipitation EMSA Supershift

2. Techniques to know to understand signal transduction • __________- Detect DNA only • ___________- Detect RNA • ___________- Detect RNA of ____ of expressed genes • ________ ( Reverse transcription polymerase chain reaction- to detect RNA) • ________________________ Detect protein • _______________- Detect proteins in situ • _________- protein-DNA interactions • ________– Protein-protein interactions • ______________- Protein-protein interactions

3. 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

4. RNA detection 1. Southern analysis- to determine if gene is present/defective 2. Northern transfer and analysis 3 2 1 3 2 1 1. Northern transfer to _____ membrane 28S Ras detected 18S 2. Hybridize with Ras _____ probe 3. Wash and expose to _____ Stained gel Film exposed to probed membrane

5. 3. Microarrays- monitor expression of several genes at once Genome-wide analysis- yeast

6. RNA detection 4. RT-Polymerase Chain Reaction (RT-PCR) One copy mRNA Fig. 4.17 many copies DNA

7. 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

8. Components of a Western analysis Protein detection 5. Western Analysis Polyacrylamide gel elecrophoresis, transfer to nylon membrane 1. Protein separation Alkaline peroxidase 2. Detection Primary Ab (Rabbit Anti-Ras) Secondary Ab (Anti-rabbit Ab) RAS If use antibody to phospho-Ras, then detect slower migrating phopho-Ras Ras protein

9. 5. Western Analysis- Example After Before Probe for specific protein using antibodies Onto nylon membrane PAGE

10. 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 ___________________

11. 6. Western Analysis 6. Immunostaining Detect protein inside of a cell? Enzyme or __________ molecule __________ Ab (Rabbit Anti-Ras) __________ Ab (Anti-rabbit Ab) RAS Cell

12. 6. Immunostaining- example 1 GP73 in human tissues Colon Lung Kidney Prostate GCH Liver Liver Immunohistochemistry Kladney et. al. Gene 249:53 2000

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

14. 7. EMSA – Detect DNA –protein complex 7. EMSA – Detects DNA–protein interaction – LPS+CHx – Ø – CHx – TNF – LPS Cell lysate + ____________ DNA NF-kB DNA-protein complex Commonly used to identify ___________ factor (SMAD, STAT, NF-kB, etc.) binding to ________________ Unbound DNA

15. 8. Supershift – “An EMSA with antibody added” LPS +CHx C CHx LPS U - - - - - + + + Cell lysate + Radiolabeled DNA + _________ to protein of interest Anti-NF-kB supershift NF-kB Used to 1. Verifies _______ of protein in DNA/protein complex 2. Identify ______ ________ in the DNA-protein complex NF-kB

16. 9. Co-immunopreciptiation- Protein-protein interactions Cell Ras Grb2 RasGEF receptor Do these interact?? Lyse cell Primary Ab (Rabbit Anti-RasGEF) Immunoprecipitate with anti-RasGEF Ab RasGEF PAGE Transfer to membrane Probe with anti- Grb2 antibody and secondary antibody Grb2 protein Only detect Grb 2 only if it is binding to RasGEF in cells