Preparation of Protein Lysates from Cells
From Bridges Lab Protocols
Revision as of 18:28, 15 December 2017 by Iharvey (Talk | contribs) (Created page with "==Materials== *RIPA Buffer (see RIPA) or other Lysis buffer. Add protease inhibitors. *Cells (fresh or frozen) ==Protocol== #Cool centrifuge to 4C #If cells...")
Materials
- RIPA Buffer (see RIPA) or other Lysis buffer. Add protease inhibitors.
- Cells (fresh or frozen)
Protocol
- Cool centrifuge to 4C
- If cells are not already frozen in buffer, add ~400ul RIPA plus PI while keeping cells on ice
- Scrape cells and transfer to cold micro centrifuge tube on ice
- Incubate on ice for 15 minutes
- Centrifuge at 14 000 RPM at 4C for 10 min
- Remove supernatant to clean tube. If lysing fat, try to avoid the floating fat cake. If necessary respin to clarify
- Follow Protein Lysate instructions for Bradford Assay (see Bradford Assay)
- Prepare samples for gels by adding 800 ug protein to a final volume of 200 uL of lysis buffer. Add 200 uL of 2X loading buffer with B-ME to each lyaste. This will generate a 2 mg/mL protein solution in SDS Loading Buffer
- Heat samples with loading buffer at 95C for 5 mins
- Snap freeze remaining clarified lysate and SDS-PAGE lysates and store at -80
