Primary tissues are valuable tools for the study of intracellular and extracellular markers which characterize disease states. We have developed a protocol for rapid isolation of cytokines and signaling molecules from intact tissue. This method is for total protein extraction and makes use of a non-abrasive tissue extraction reagent. Tissue samples as little as 10 mg may be extracted using this protocol. This method is sensitive and allows for the detection of disease-associated fluctuations of biomarkers. This is an effective system for the extraction of proteins from a variety of tissue types. Heart, lung, kidney, spleen, brain, liver, thymus, and smooth muscle tissues have all been successfully extracted with this protocol. The prepared extracts are compatible with a variety of immunoassays, such as ELISA and Invitrogen Luminex platform, as well as with all conventional protein assays. Our extraction method is inexpensive, versatile, and can be completed in less than 15 minutes. We offer a Cell Extraction Buffer for total protein extractions from various tissue sample types.
Prepare 1X Cell Lysis Buffer (Cell Extraction Buffer) using the following formulation:
Cell extraction buffer formulation
This Cell Extraction Buffer is available—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 (Cat. No. 78429)
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 Halt Protease Inhibitor Cocktail (Cat. No. 78429), reconstituted to 1X, and adding 50 μL per 5 mL Cell Extraction Buffer. The stability of protease inhibitor-supplemented Cell Extraction Buffer is 24 hours at 4°C.
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.
BioSource C-070276 1107 1-Jan-2007