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Total protein extraction kit (strong) 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.
Kit Components:
规格 | 储存条件 | |
裂解液 | 100ml | 2-8℃ |
磷酸酶抑制剂(100x) | 1ml | -20℃ |
蛋白酶抑制剂(100x) | 1ml | -20℃ |
PMSF(100x) | 1ml | -20℃ |
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.
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1.组织一定要用新鲜的吗?从-80冰箱拿出来的样可以用于保存吗?
2.取样之后来不及马上就提蛋白,需要怎么处理?可不可以加入裂解液,磷酸酶抑制剂、蛋白酶抑制剂和PMSF后放在-80℃保存着?如果可以的话,可以保存多久?2022-07-10 16:59:26
Administrator冻存的组织也可以用于蛋白的提取;取样后不能马上提取蛋白,组织直接-80保存,一般可保存6个月