What is the principle of RIPA cracking liquid

RIPA lysate (RIPA Lysis Buffer) has a strong effect on the cell membrane, cytoplasm and nuclear components of animal cells. The protein samples obtained from RIPA lysate can be used for routine Western,IP and so on. The principle of RIPA lysate is as follows(http://www.hnhbsj.com).

RIPA is mainly a soluble protein extracted from animal tissues and cells. RIPA tissue / cell lysates use surfactants to break up cell membranes (including nuclear membranes). If RIPA is found to have precipitation, please leave it at room temperature for half an hour or water bath at room temperature to dissolve it.

According to the amount used, the final concentration of PMSF was 1 mm by adding 10ul PMSF, per 1mlRIPA. Mix and set aside (PMSF now added).

1. Sample pretreatment(http://www.hnhbsj.com):
A) for adherent cells: remove the culture medium and wash it with PBS, saline or serum-free medium. According to the number of cells in each hole of the 6 hole plate, the 150-250ul lytic solution was added. With the gun blow several times, the lytic liquid and the cell fully contact.
B) for suspension cells: centrifugal collection of cells, with the fingers of the cells to bounce the force. According to the amount of cells in each hole of the 6 hole plate, add the lytic solution to 150-250 ul pyrolysis liquid, and then use the fingers to fully crack the cells. There should be no obvious cell precipitation after full lysis. If the number of cells is large, must be packed into 50-1 million cells / tube, and then split.
C) for tissue samples: cut the tissue into small pieces. According to the ratio of 150 to 250 ul pyrolysis solution per 20mg tissue, the lytic solution was added. More lytic fluids can be added if the cleavage is not sufficient, and if high concentrations of protein samples are needed, the amount of the lytic solution can be reduced appropriately.
Use glass homogenizer to homogenize until fully cracked.

2, reprocessing:
The dissociated samples were centrifuged for 3-5 minutes from 10000-14000g for 3 to 5 minutes. The supernatant could be used for further operation of PAGE,Western and immunoprecipitation.

Note(http://www.hnhbsj.com):
The strong cleavage solution can extract the nucleoprotein, but at the same time, it releases the genome together, causing the cell lysis fluid to be sticky, so you can directly add the protein sample buffer, boil and centrifuge. Centrifugation followed by direct sample electrophoresis; If you want to determine the concentration, add a small amount of SDS (1%), boiling centrifugation concentration. The protein extracted by this series of protein extraction reagents is not suitable to use Bradford protein concentration determination kit because it contains detergent. Please choose BCA or Lowry method to detect protein concentration.

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