Collection of Proteins and RNA Post- Stimulation

Collection of Proteins and RNA Post- Stimulation

Collecting proteins at each stimulation time point

1. Warm solutions needed (PBS, lysis buffer)
2. Clean cell scrapers in DI water, place in hood
3. Collect ice for the samples
4. Label three AUTOCLAVED 1.7 ml tubes for each condition (PBS 1-2-3 and agrin 1-2-3) include cell type, RNA or AG, time point tube number. Label on side and lid
5. Take cells into hood and remove old media with pipette tips and vacuum *do one treatment at a time*
6. Wash with 2 ml PBS for 10 seconds and remove
7. Add 300 ul of lysis buffer per well
8. Detach cells by scraping (10-15 times)
9. Take the 300 ul of cells scraped and flush
10. Put into the labeled tube and put on ice

Collecting RNA at each stimulation time point

1. Warm solutions needed (PBS)
2. Collect trizol from white fridge. Trizol degrades cells but keeps the RNA
3. Clean cell scrapers in DI water, place in hood
4. Collect ice for the samples (keep trizol on ice)
5. Label three AUTOCLAVED 1.7 ml tubes for each condition (PBS 1-2-3 and agrin 1-2-3) include cell type, RNA or AG, time point tube number. Label on side and lid
6. Take cells into hood and remove old media with pipette tips and vacuum *do one treatment at a time*
7. Wash with 2 ml PBS for 10 seconds and remove
8. Add 1 ml of trizol per well
9. Detach cells by scraping (10-15 times)
10. Take the 1 ml of cells scraped and flush
11. Put into the labeled tube and put on ice
12. Wash old plate with 1 ml of PBS for smell and remove. Trash plate.