Cell Extraction Protocol
Cell extraction buffer formulation
* 100 mM Tris, pH 7.4
* 2 mM Na3VO4
* 100 mM NaCl
* 1% Triton X-100
* 1 mM EDTA
* 10% glycerol
* 1 mM EGTA
* 0.1% SDS
* 1 mM NaF
* 0.5% deoxycholate
* 20 mM Na4P2O7
This Cell Extraction Buffer is available from Invitrogen (Cat. no. FNN0011).
This Cell Extraction Buffer may be apportioned into 1x aliquots in microcentrifuge tubes and stored at –20°C until ready for use. Thaw on ice prior to extracting cells.
Additional Reagents Needed:
1 mM PMSF
Protease Inhibitor Cocktail, Sigma (Cat. no. P-2714)
This Cell Extraction Buffer must be supplemented with 1 mM PMSF (not included) and Protease Inhibitor Cocktail (not included) just prior to use to make Complete Cell Extraction Buffer. Addition of the Protease Inhibitor Cocktail and PMSF is necessary to inhibit proteolysis in cell extracts. For the PMSF addition, we recommend making a 0.3 M stock in DMSO, and adding sufficient volume for a final concentration of 1 mM (i.e., 17 μl per 5 ml Cell Extraction Buffer). PMSF is very unstable and must be added just prior to use, even if added previously. For the Protease Inhibitor Cocktail addition, we recommend Sigma (Cat. no. P-2714), reconstituted according to the manufacturer’s instructions, and adding 250 μl per 5 ml Cell Extraction Buffer. The stability of protease inhibitor-supplemented Cell Extraction Buffer is 24 hours at 4°C.
- Following the end of the desired cell culture time, pipette medium into a microcentrifuge tube and
- immediately put on ice.
- Centrifuge at 1,400 rpm for 1 minute.
- Remove supernatant fluid and aliquot into a clean microcentrifuge tube.
- Store at –80°C until ready for use in ELISA.
This method can be used to produce relatively large quantities of cell extracts with each of the stimulation regimes studied. The wash steps included in this procedure help to minimize medium components in the cell extracts.
- Estimate cell density: Suspension Cells: Enumerate suspension cells by counting in a hemacytometer. Adherent Cells: Estimate cell density by visual inspection under a microscope. Confluence levels of 70–80% are found to be optimal for many signal transduction studies.
- Stimulate cells as desired.
- Transfer the cells into clean 15 ml conical tubes: Suspension Cells: Aliquot the desired number of cells in medium into clean 15 ml conical tubes. Adherent Cells: Remove the cells from their vessel by scraping. Transfer the medium containing the detached cells into clean 15 ml conical tubes.
- Collect the cells by centrifugation at 300 x g for 7 minutes.
- Aspirate the medium.
- Resuspend the pellet in ice-cold PBS.
- Collect the cells by centrifugation at 300 x g for 7 minutes at 4°C.
- Aspirate the PBS.
- Lyse the cells by pipetting Complete Cell Extraction Buffer into each tube. We recommend using 1 ml of Complete Cell Extraction Buffer per 108 cells. It is important to note that this value may require optimization for each specific application.
- Transfer the lysates to clean microcentrifuge tubes.
- Vortex the mixture, then incubate the mixture on ice for 30 minutes, with occasional vortexing.
- Clarify the lysates by centrifugation at 14,000 rpm (13,000 x g) at 4°C for 10 minutes.
- Transfer the clarified cell extracts to clean microcentrifuge tubes.
- The clarified cell extracts should be stored at –80°C until ready for analysis. Avoid repeated freeze-thaw cycles. In preparation for performing the assay, allow the samples to thaw on ice. Mix well prior to analysis.
- Determine protein concentration using a suitable method, such as the Quant-iT™ Protein Quantitation Kit (Cat. no. Q33210). Cell extracts prepared by this method routinely have a protein concentration between 1 and 10 mg/ml.
- Certain analytes require a sample treatment step. Please refer to the analyte specific protocol for details on sample treatment recommendations.
- All cell extracts require dilution by a factor of at least 1:10 in Standard Diluent Buffer before analysis with Invitrogen™ kits.