Transport temperature: normal temperature transport

This product is a highly efficient tissue cell lysate. It is mainly used to extract soluble proteins from animal tissues and animal cells. Tissue Live cells This product is equipped with a PMSF (0.3ml/1.5ml), the PMSF should be stored at -20℃.

Instructions for use

If RIPA is found to have precipitation, please let it sit at room temperature for half an hour or in a room temperature water bath to dissolve the precipitation.

According to the amount used, 10ul PMSF is added to every 1ml RIPA to make the final concentration of PMSF 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. The lysate was added at the ratio of 150-250 ul lysate per cell volume of the 6-well plate. Blow the gun a few times to make full contact between the lysate and the cells.

b) For suspended cells: Collect the cells centrifugally and use your fingers to forcefully flick the cells apart. Add the lysate at the ratio of 150-250 ul per cell volume of the 6-well plate, and then flick with your finger to fully lysate 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 ul lysate per 20mg 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:

Centrifuge 10000-14000g of the cracked sample for 3-5 minutes, take the supernatant, and then perform subsequent PAGE, Western and immunoprecipitation operations.

Note:

This reagent is a strong lysate, which can extract nuclear protein, but at the same time of extracting nuclear protein, the genome will also be released, resulting in a thick cell lysate. At this time, the protein sample buffer can be directly added, boiled and centrifuged, and the sample is directly loaded after centrifugation. To determine the concentration, add a small amount of SDS (1%), boil and centrifuge the concentration. The protein extracted by this series of protein extraction reagents contains stain remover, so it is not suitable for Bradford protein concentration determination kit. Please choose BCA method or Lowry method to detect protein concentration.

If a protein that is particularly tightly bound to the genome needs to be detected, the goo can be broken up by ultrasound treatment, then the supernatant can be centrifuged for subsequent experiments.

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