Phosphorylated protein extraction kit is used to extract total protein from mammalian tissues and cells. The lysate in the kit contains protease inhibitors and phosphatase inhibitors, which have a strong effect and can quickly obtain total protein. It can be used for basic research experiments such as western blot experiments. This product is used for scientific research only.

Operation method:

1. Extraction of total tissue protein

① 10ul phosphatase inhibitors, protease inhibitors and PMSF were added to 1ml of cold lysate; Mix well and set aside on ice.

(2) Weigh 0.1g of fresh tissue, place it at the inlet buffer of the glass homogenizer, cut the tissue block as much as possible with ophthalmic scissors, then add 0.5-1ml of newly configured cracking liquid, and grind until there is no obvious tissue block, this process should be careful to operate on the ice;

③ The homogenate was transferred into 1.5ml EP tube and centrifuged at 4℃ 12000g for 30min;

④ Absorb the supernatant into the new tube;

⑤ Protein quantification or denaturation for protein experiments. (The extracted protein is recommended to be stored at -80℃, ready to use, to avoid repeated freezing and thawing, to avoid long-term storage.)

2. Extraction of total cell protein

① The amount of lysate: 10⁷ cells required 1ml lysate;

② Adherent cells: Discard the medium, wash twice with cold PBS, discard PBS, and then add the calculated cell lysate; The cells were scraped off with a cell scraper on the ice, and the scraped cell lysate was transferred into the EP tube for reverse cleavage for 20-30min.

③ Suspension cells: Centrifuge cells at 4℃ 400g, wash cells twice with cold PBS, add lysate according to the number of cells, swirl for 10s, place on ice for cracking for 10min, repeat 3-4 times;

④ After lysis, the cell lysate was centrifuged at 4℃ 12000g for 30min;

⑤ Transfer the supernatant into the new EP tube;

⑥ Protein quantification or denaturation for protein experiments. (The extracted protein is recommended to be stored at -80℃, ready to use, to avoid repeated freezing and thawing, to avoid long-term storage.)

Note:

1. During the experimental process, all reagents need to be pre-cooled or melted to ensure a low temperature environment during the operation;

2, PSMF (toxic) is used now, because PMSF will degrade rapidly in aqueous solution.

Note：Product information may be optimized and upgraded. Please refer to the actual label information for accuracy.