General Notes
Experimental Protocol for Adherent Cells 1. Prepare Lysis Buffer: Just before use, make IntactProtein™ lysis buffer by adding 2 microliters of Reagent A to 1 milliliter of Reagent B. Vortex thoroughly and place on ice. 2. Wash Cells: Remove the culture medium and wash cells twice with ice-cold PBS. 3. Add Lysis Buffer: Place the culture dish/plate on ice or in ice water. Add 1 milliliter of the premixed lysis buffer per 5 million cells (e.g., 300 microliters for a 35?mm dish containing 1 million cells). Keep on ice for 5 minutes, swirling occasionally to ensure complete coverage. 4. Collect Lysate: Scrape cells gently with a clean plastic scraper and transfer the lysate to a centrifuge tube. 5. Enhance Lysis: Vortex the lysates thoroughly (3×10 seconds), then keep on ice for another 10 minutes to complete lysis. 6. Heat Lysates: Incubate the lysates at 95°C for 5 minutes. 7. Cool Lysates: Place on ice for 3 minutes. 8. Centrifuge: Spin at 13,000 times gravity for 5 minutes at 4°C. 9. Measure Protein: Determine protein concentration using a NanoDrop spectrophotometer or SDS-compatible protein assay. Store or Use: Store lysates at –20°C or proceed immediately with analysis. Experimental Protocol for Suspension Cells: 1. Prepare Lysis Buffer: As described in Step 1 of the adherent cell protocol. 2. Wash Cells: Centrifuge cells at 300?g for 5?min, resuspend in 10?mL ice-cold PBS, centrifuge again, discard PBS, and resuspend in the remaining PBS by pipetting. 3. Lyse Cells: Add 1?mL of premixed lysis buffer per 5×106 cells. Mix by pipetting and incubate on ice for 5?min. Proceed: Continue with Steps 5–10 of the adherent cell protocol. Experimental Protocol for Tissues: 1. Prepare Lysis Buffer: As described in Step 1 of the adherent cell protocol. 2. Grind Tissue: Using liquid nitrogen, grind tissue into a fine powder with a mortar and pestle. 3. Add Lysis Buffer: Mix frozen tissue powder with premixed lysis buffer at 1?g tissue : 3?mL buffer. 4. Homogenize: Homogenize according to the manufacturer’s instructions. Tip: Keep tubes on ice to prevent heat buildup during homogenization. 5. Incubate: Keep homogenized samples on ice for at least 15?min for complete lysis. Tip: If processing multiple samples, keep all on ice until the last is homogenized. 6. Centrifuge: After the last sample has lysed for 15?min, centrifuge at 13,000?g for 10?min at 4?°C. Transfer the supernatant to a clean tube. Proceed: Follow Steps 6–10 of the adherent cell protocol.
