The non-denatured tissue/cell lysis buffer contains a non-ionic detergent that can lyse cells and release cytoplasmic proteins, soluble membrane proteins and nuclear proteins under non-denatured conditions. The cleavage protein products under non-denaturing conditions retain the properties and functions of the protein to the maximum extent, such as antigen-antibody binding or enzymatic activity, and are therefore suitable for immunoprecipitation. In addition, some antibodies have higher binding capacity to non-denatured proteins or can only recognize antigenic sites of non-denatured proteins, in which case non-denatured cleavage should be used.

Instructions for use:

According to the amount used, 10ul PMSF is added to every 1ml of the lysate, so that the final concentration of PMSF is 1mM. Mix well and set aside (PMSF add now).

1. Sample pretreatment:

a) For adherent cells: Remove the culture medium and wash it again with PBS, normal saline or serum-free culture medium. Add the cracking liquid at the ratio of 150-250ul per well of the 6-well plate. Blow several times with the gun to make the lysate and the cells fully contact, and place the lysate on the ice for 5-10 minutes.

b) For suspended cells: Collect the cells centrifugally and use your fingers to forcefully flick the cells apart. The lysate was added according to the ratio of 150-250 microliters of lysate per cell in the 6-well plate, and the lysate was placed on the ice for 5-10 minutes. You can flick it with your fingers to fully lyse the cells. There should be no obvious cell precipitation after full lysis. If the number of cells is large, it must be divided into 500,000 to 1 million cells/tubes, and then cracked.

c) For tissue samples: Cut the tissue into fine fragments. The lysate is added at a ratio of 150-250 microliters per 20 mg of tissue. (If the cleavage is insufficient, more lysate can be appropriately added, and if a high concentration of protein samples is required, the amount of lysate can be appropriately reduced). Homogenize with a glass homogenizer until fully cracked.

2. Post-processing:

After full cracking, the cracked sample was centrifuged 10,000-14000g for 3-5 minutes, and the supernatant was taken, and subsequent PAGE, Western, immunoprecipitation, immunocoprecipitation and other operations could be carried out.

Note:

In order to achieve the best use effect, try to avoid too much repeated freezing and thawing. Can be used after appropriate packaging. All the steps of cracking the sample are carried out on ice or at 4 ℃. The cracking time can be optimized according to the experimental requirements.

The protein sample obtained from non-denatured protein lysate can be determined by BCA protein concentration determination kit. Due to the high concentration of Triton X-100 and other interfering substances, the protein concentration of the samples obtained from the lysate cannot be determined by the Bradford method.

For your safety and health, please wear a lab coat and disposable gloves.

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