Description of active protein extraction method

Active Protein Extraction Kit Store at -20°C for 12 monthsFor Research Use Only 1. Kit Description This kit is used to extract active whole protein from mammalian tissues and cultured cells, inhibited by protease inhibitors and phosphatase. The Lysis Buffer homogenate lyses the tissue or cells, the action is mild, and the extraction process is simple and efficient. The obtained whole protein can be used for subsequent studies such as Western Blot, co-immunoprecipitation and determination of enzyme activity, but can not be used for the study of protein kinase and phosphatase co-precipitation, because the inhibition of these two enzymes is included in the kit. Agent. Application: It can be used for subsequent studies such as Western Blot, co-immunoprecipitation and enzyme activity determination. II. Kit component KGP105050 tests KGP10100100 tests Storage temperature Lysis Buffer 50mL 100mL 2-8°C phosphatase inhibitor 500μL 1000μL -20°C Protease inhibitor 50μL 100μL PMSF (100mM) 500μL 1000μL -20 °C, protected from light III, operation step I extraction of solid tissue protein 1. Add 10 μL of phosphatase inhibitor, 1 μL of protease inhibitor and 10 μL of PMSF per mL of cold Lysis Buffer and mix. Keep it on ice for a few minutes. 2. Each 100 mg of solid tissue was placed in a Petri dish, surgically cut into small pieces of about 3 mm × 3 mm, 0.5 to 1 mL of cold Lysis Buffer was added, and the glass homogenizer was manually homogenized 15 times, paying attention to low temperature operation; The tissue homogenate was transferred to a 1.5 mL pre-cooled centrifuge tube, centrifuged at 10,000 rpm, and centrifuged at 4 ° C for 5 min; Transfer the supernatant to a new pre-cooled centrifuge tube, which is a whole protein extract, protein quantification (Bradford method, BCA method); Store in -70 ° C in separate parts to avoid repeated freezing and thawing. II culture cell protein extraction The adherent cultured cells were aspirated, and then washed twice with cold PBS added to a 10 mL/150 mm culture plate, shaking each time several times to remove the culture solution as much as possible; Suspension cultured cells or adherent cells scraped with cell scrapers, transfer the cells and culture medium to a centrifuge tube, 1000 rpm for 10 min, and then use 10 mL/150 mm culture plate of cold PBS, 1000 rpm/separation Wash the heart twice for 5 min; 3. Add 10 μL of phosphatase inhibitor per mL of cold Lysis Buffer, 1 μL of protease inhibitor and 10 μL of PMSF (user-adapted: concentration 100 mM, dissolved in isopropanol), and mix. Keep it on ice for a few minutes. 4. Add the above-mentioned prepared cold Lysis Buffer to the cells of the above culture plate or centrifuge tube, and add the amount as shown in the following table: Number of cells Lysis Buffer Addition amount 107 0.5 mL to 1 mL 5 × 106 0.2 mL to 0.5 mL 5. In the cold room or on ice, the adherent cells are scraped off with cell scrapers and transferred to a new pre-cooled centrifuge tube with Lysis Buffer; the adherent cells scraped before suspension or lysis are all transferred to the new Lysis Buffer. Pre-cooled centrifuge tube, 6. Place on a 4 ° C shaker platform and gently shake for 15 min; Centrifuge at 14,000 rpm for 15 min at 4 ° C, and take the supernatant as a whole protein extract, protein quantification (Bradford method, BCA method); Store in -70 ° C in separate parts to avoid repeated freezing and thawing. 4. Precautions • All utensils and reagents that come into contact with the sample should be pre-cooled. ? After extracting the protein, if necessary, dialyzed to remove the surfactant

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