SAMPLE PREP

  1. Harvest cells by adding 1ml ice-cold PBS and scraping (pre-cool cell scrapers and microtubes). Spin 5min in mini centrifuge
  2. Prepare lysis buffer:
    1. 1ml Ripa buffer (stored at 4°C)
    2. 10ul protease inhibitor (PI) (100x)
    3. 10ul phosphatase inhibitor
    4. 1ul DTT
  3. Thaw cells in ice and spin in mini centrifuge or pre-cool centrifuge for 1min. Remove ALL liquid using pipette (important for determining protein concentration)
  4. Resuspend cells in 20-100ul lysis buffer (depending on pellet size), incubate on ice for 30min, start Fast Cool on centrifuge
  5. Spin 10min at maximum speed at 4°C. Prepare Bradford reagent (stored at 4°C) (5x to 1x with H2O) while samples are spinning
  6. Transfer the same column of supernatant to new tube
  7. Add 1ul supernatant to 1ml Brad reagent, invert, wait 2min. Blank w/ 1ul lysis buffer
  8. Aliquot 100ul/well into clear bottom 96-well plate. Do each sample in triplicate
  9. Run “Bradford.prt” program on plate reader to determine 595nm absorbance values
  10. Open “Bradford assay template.xls” and input 595abs, sample volume, and desired normalized concentration. Print out output
  11. Add additional lysis buffer to normalize each sample in the volumes indicated by the excel sheet
  12. Prepare 1ml sample buffer by
    1. 950ul Laemmli
    2. 50ul beta-ME
  13. Add sample buffer in volumes indicated by excel sheet. All samples should be the same concentration
  14. Lock cap and boil on heating block for 5min

RUNNING GEL AND TRANSFERRING

  1. Prepare Bolt running buffer (if not already prepared) by diluting 20x concentrate with diH2O
  2. Set up gel box and add running buffer over the top of metal electrode