Description
N748603Component100mL500mLStorageN 748603ANP-40 cracking solution100mL500mL2-8℃N748603B50x phosphatase inhibitor 2x1mL10x1mL-20℃. Avoid freeze/thaw cycle.N748603C100xPMSF 1mL5x1mL-20℃. Avoid freeze/thaw cycle.NP-40 Lysis Buffer is a relatively mild cell tissue lysis buffer, and the protein samples obtained from its lysis can be used for conventional Western, IP, and co IP assays. The protein sample obtained by lysing with NP-40 lysate can be measured for protein concentration using the BCA protein concentration assay kit. Due to the high concentration of descaling agents, the Bradford method cannot be used to determine the protein concentration of the sample obtained from the lysis of this lysate.Precautions：1. To achieve the best usage effect, it can be appropriately packaged and stored at -20 ℃ to avoid excessive repeated freezing and thawing;2. All steps of sample cracking must be carried out on ice or at 2-8 ℃;3. For your own safety, please take protective measures such as wearing lab coats and gloves before using the reagents.Instructions for Use：1. Reagent preparationTake an appropriate amount of lysis buffer and add the protease inhibitor complex and/or phosphatase inhibitor complex to the lysis buffer at a ratio of 1:50 within a few minutes before use, or use 100 mM PMSF and make the final concentration of PMSF 1 mM.2. Cell/tissue lysisFor adherent cells: Remove the culture medium and wash once with PBS, physiological saline, or serum-free culture medium. Add lysis buffer in a ratio of 150-250 uL per well to a 6-well plate. Blow with a gun several times to ensure full contact between the lysate and the cells. Usually, after 1-2 seconds of contact with the cell lysate, the cell will be lysed. If used for ChIP, it needs to be further cracked on an ice bath for 10 minutes after initial cracking; For suspended cells: Collect cells by centrifugation and use your fingers to forcefully scatter them. Add lysis buffer at a ratio of 150-250uL per well of cells in a 6-well plate. Gently flick with your fingers to fully lyse the cells. If there are a large number of cells, they must be divided into 500000 to 1 million cells/tube and then lysed. After sufficient lysis, there should be no obvious cell precipitation. If used for ChIP, it needs to be further cracked on an ice bath for 10 minutes after initial cracking. For tissue samples: Cut the tissue into fragments using a tissue cutter, add 150-250uL of lysis buffer per 20mg of tissue, and homogenize with a glass homogenizer until fully lysed. If used for ChIP, it needs to be further cracked on an ice bath for 10 minutes after initial cracking.3. Collection of protein samplesAfter sufficient lysis, centrifuge at 10000-14000g for 3-5 minutes, take the supernatant, and proceed with subsequent PAGE, Western blot, and ChIP operations