NP-40 Lysates

Nonidet P-40 (also referred to as NP-40) is a widely used non-ionic scaling agent that helps to remove unwanted deposits. An NP-40 Lysis Buffer is a mild pyrolysis liquid tissue that contains key ingredients such as 50mm Tris-HCl (pH 7.4), 150mm NaCl, NP-40, and 1% sodium pyrophosphate, sodium vanadate, sodium fluoride, EDTA, and aprotinin inhibitor. This unique blend of chemicals effectively inhibits protein degradation, making it an ideal tool for obtaining protein samples that can be used in various experiments such as Western Blot, IP, and Co-IP.
When using NP-40 Lysis Buffer, the protein samples collected can be analyzed to determine their total protein concentration using the enhanced BCA protein concentration determination kit. It is important to note that due to the high concentration of detergent in this product, the Bradford method is not suitable for determining total protein concentration.
Overall, NP-40 is a powerful and essential tool for any researcher working with protein samples, as it allows for the effective removal of unwanted scaling and inhibits protein degradation.

A Cultured cell samples
(2) To ensure complete mixing, it is important to fully dissolve the NP-40 pyrolysis fluid before proceeding. Once dissolved, take an appropriate amount of the pyrolysis solution and introduce PMSF (molecular weight: 174.2g/mol) into it just a few minutes before usage. The aim is to achieve a final concentration of 1mM. It is crucial to follow this protocol to guarantee consistent results and optimal performance.
To extract protein from adherent cells, start by removing the culture medium and rinsing with a suitable solution such as PBS, normal saline, or serum-free culture medium. If serum proteins do not interfere with the experiment, the washing step can be skipped. Next, add 150-250ul of lysate to each well of the 6-well plate and ensure proper contact between the lysate and cells by applying a few gentle blows with a gun. The lysis process should last between 15 to 30 minutes, and usually, within 1 to 3 seconds of the lysate's contact with the cell, the cell will be lysed. Overall, the carefully planned steps guarantee optimal protein extraction from adherent cells.
To suspend the cells, start by collecting them through centrifugation and gently scatter them with your fingers. Once that's done, add the appropriate amount of lysate to each well of the six-well plate, maintaining a ratio of 150-250ul lysate to cells. After adding the lysate, make sure to flick the plate with your finger to achieve full cell lysis. It's important to note that there should be no significant cell precipitation observed after complete lysis. In case there is a large number of cells, it is necessary to divide them into tubes containing 500 to 1 million cells and then proceed with the cell lysis process.
Following a full lysis of the cellular materials, the resulting supernatant was separated through a centrifugation process. This involved the application of force, in the range of 10,000-14,000g for approximately 3-5 minutes, to effectively extract the desired materials for downstream applications. Subsequent experiments such as polyacrylamide gel electrophoresis (PAGE), western blotting, and immunoprecipitation could then be conducted with the prepared supernatant.
To ensure optimal results, it is recommended to add 150ul of lysate to each well in a 6-well plate. However, if the cell density is particularly high, it may be necessary to increase the lysate dosage to 200ul or even 250ul to ensure complete coverage. It is important to carefully consider the appropriate lysate dosage for your specific experimental conditions to achieve the most accurate and reliable results. By following these guidelines, you can ensure that your lysate is used efficiently and effectively in your research.
B Tissue sample
(1) Cut the tissue into small pieces.
(2) Fully dissolve NP-40 pyrolysis fluid, and then mix.Take an appropriate amount of pyrolysis solution and add PMSF (mW: 174.2g /mol) a few minutes before use, so that the final concentration is 1mM.
(3) According to the ratio of adding 150-250ul pyrolysis solution per 20mg tissue, increase the amount.[Note: If cleavage is insufficient, dosage can be added appropriately. If high concentration protein sample is needed, dosage can be reduced appropriately.]
(4) Use glass homogenizer to homogenate until fully cracking, and this process should be controlled within 1-2 minutes as far as possible to reduce protein degradation.
(5) After full lysis, the supernatant was extracted by centrifugation at 10,000-14,000g for 3-5 minutes, and the subsequent PAGE, WB, immunoprecipitation and other experiments could be performed.
(6) If the tissue sample itself is very small, it can be cut properly and then directly added to the cracking liquid for cracking. The sample can be fully cracked through the high-strength whirlpool mixer, and then the supernatant can be extracted by the same centrifugation for subsequent experiments.The advantage of direct cracking is that it is more convenient and does not require the use of a homogenizer, but the disadvantage is that the cracking is not as sufficient as the use of a homogenizer.

1. All steps of cracking samples should be carried out on ice or at 4° C.2. Different cells may need to add different amount of lytic fluid, which requires conditional screening according to specific conditions;3. For your own safety, please do a good job of protection before using the reagent, such as wearing laboratory clothes and gloves.

Store at -20° C

NP-40 Lysis Buffer