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Cell Lysis Protocol: *All steps should be done on ice. 1.

Thaw inhibitors for the cell lysis buffer (Na3VO4 is at -20 C, and the Phosphatase Cocktail is at 4 C in DMSO). 2. Label tubes for lysates so they are ready once the lysate is collected. 3. Obtain the necessary amount of GTP Lysis Buffer Stock for what you are doing and add the following: a. 20L/mL of PMSF b. 20L/mL of Na3VO4 c. 10L/mL of Aprotinin d. 10L/mL of NaF e. 10L/mL of Protease Cocktail f. 10L/mL of Phosphatase Cocktail 4. Aspirate the media from the cells. 5. Wash the cells 2x with ice cold dPBS, aspirating the wash off after each. 6. Tilt the plates and allow any excess PBS to accumulate on one side to be aspirated. 7. Add the appropriate amount of cell lysis buffer. 8. Scrape the cells with a cell scraper thoroughly. 9. Collect the cell lysate and place it in a labeled tube. 10. Allow the lysate to sit on ice for 30 minutes. 11. Spin the cells in the cold room at 12,000xg for 5 minutes. 12. Transfer the supernatant to a clean tube (the pelleted debris can be discarded). 13. The lysates can now be assayed for protein amounts, or can be stored at -80 C for future use.

Protein Assay Protocol: 1. If you are thawing previously frozen samples on ice, be sure the thaw is complete before proceeding (this can take up to 1.5 hours if the sample had a large volume). 2. Get protein standards out to thaw. 3. To the 96-well assay plates, add a 1g, 2.5g, 5g, 10g and 20g standard in triplicate (I use a 1g/L stock and add 20L for the 20g standard, 10L of stock + 10L of dH2O for the 10g standard, etc.), in addition to a blank with 20L of dH2O. 4. Add 1L of your samples + 19L of dH2O in triplicate to empty wells (you can use 5, 10 or 20L as well, but make sure to account for the dilution factor). 5. Add 25L of the Protein Assay Reagent A to each well with a sample, standard or blank. 6. Add 200L of the Protein Assay Reagent B to each well that got Reagent A. 7. Allow the plate to sit for 5-30 minutes at room temperature. 8. Read the plate at OD-650nM in the Schooley Lab across the hall (please ask someone to show you the first time you do this protocol as the instrument is very old and has some nuances). This machine will use your standards to determine the protein concentrations of your samples for you. The only thing to keep in mind is the reading is for g/L, and you need to account for any dilution you made.