Lysates (Without Protease / Phosphatase Inhibitors)

Western and IP cell lysates are commonly used for lysing cells without denaturing the proteins. These lysates are widely used in PAGE, Western, immunoprecipitation, and immunoblotting techniques. Specific inhibitors can be added to the lysates depending on the intended purpose. The primary components of Western and IP cell lysates include Tris-HCl, NaCl, and a descaling agent. These lysates effectively prevent protein degradation and maintain native protein interactions.
Before using the lysate for detecting enzyme activity or biological small molecules, it is crucial to test whether the dilution of the standard substance with the lysate affects the standard curve. If not, the lysate can be utilized for such applications. For determining protein concentration in samples, the BCA protein concentration assay kit is commonly used as the Bradford method is not applicable due to the presence of detergents and other interfering substances in the lysate.
Therefore, the Western and IP cell lysates provide a non-denaturing approach for lysing cells to maintain protein interactions and prevent protein degradation. These lysates are versatile and can be used in multiple techniques for a wide range of applications.

1. For cultured cell samples
To ensure accurate results, it is important to carefully measure the lysate before adding the appropriate inhibitor or no inhibitor, depending on the specific requirements of the experiment. This step is crucial to obtaining reliable data and obtaining meaningful outcomes.
To extract proteins from adherent cells, start by discarding the culture medium and rinsing with PBS, normal saline, or serum-free culture medium (unless the serum protein does not cause interference). Next, add an appropriate amount of cracking fluid, typically 100-200uL per well of a 6-well plate, ensuring that the lysate comes into full contact with the cells. You can use a pipette to gently blow air to facilitate the interaction between the lysate and the cells. Generally, the cells will be lysed within 1-2 seconds of the lysate contact. It is important to follow these steps to obtain reliable protein samples from the adherent cell cultures.
To collect suspension cells, it is necessary to perform centrifugation followed by gently shooting the cells away using your fingers. For each well of a 6-well plate, the cells should be treated with 100~200uL of lysate. To ensure complete cell lysis, the cells can be flicked with your finger. It is important to note that after full lysis, there should be no significant cell precipitation. In cases where the cell count is high, it is recommended to divide the cells into tubes containing 5~10×105 cells each before performing lysis. Larger cell clusters pose a challenge in achieving complete lysis, while a smaller cell population is relatively easier to lyse due to better contact between the cells and the lysate fluid.
(4) After full lysis, centrifugation was conducted at 10000~14000g for 3~5min, and the supernatant was extracted, which could be used for subsequent PAGE, Western, immunoprecipitation, immunoprecipitation, enzyme or biological small molecule detection and other operations.
Note: Description of lysate dosage: Usually 100uL of lysate per well of 6-well plate is sufficient, but if the cell density is very high, the dosage of lysate can be appropriately increased to 150uL or 200uL.
2. For tissue samples
(1) Take an appropriate amount of lysate and add the appropriate inhibitor or no inhibitor according to the need.
(2) cut the tissue into small pieces.
(3) Add cracking fluid at a ratio of 100~200uL per 20mg of tissue.(If cleavage is insufficient, more cleavage solution can be added appropriately, and if high concentration protein sample is needed, the amount of cleavage solution can be reduced appropriately.)
(4) Homogenate with glass homogenizer until fully cracked.
(5) After full lysis, the supernatant was extracted by centrifugation at 10000~14000g for 3-5min, and the subsequent PAGE, Western, immunoprecipitation and immunoco-precipitation operations could be carried out.
(6) If the tissue sample itself is very small, it can be cut properly and then directly added to the cracking fluid for cracking, and the sample can be fully cracked through the strong vortex.Then the supernatant was extracted by the same centrifugation for subsequent experiments.The advantage of direct cracking is that it is more convenient and does not need to use a homogenizer, but the disadvantage is that it is not as sufficient as cracking with a homogenizer.

Store at -20° C for 1 year.

Lysis Buffer for WB/IP Assays (Without Enzyme Inhibitors)