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Do you know the method of cell lysis
Hengyuan help you easily get the experiment, to build the domestic ELISA kit leading enterprises, our company can provide tens of thousands of ELISA kits and related scientific research products, the test objects are anticoagulant whole blood, multi-fiber whole blood, a variety of animal and plant plasma (serum), to produce high quality, low price products, favored by many scientific research units. Cell lysis is an important experimental method to change the permeability and activity of cells. Today, we analyze the "method of cell lysis" and invite you to take a look!
For cultured cell samples:
1. Melt RIPA lysate and mix well. Take the appropriate amount of cracking solution, and add PMSF within a few minutes before use, so that the maximum final concentration of PMSF is 1mM.
2. For adherent cells: Remove the culture solution and wash it with PBS, normal saline or serum-free culture solution (if the protein in the serum does not interfere, you can not wash it). The lysate is added at a ratio of 150-250 microliters per well of the 6-well plate. Blow the gun a few times to make full contact between the lysate and the cells. The cells are usually lysed after 1-2 seconds of contact with the lysate.
For suspended cells: Collect the cells by centrifugation and flick the cells vigorously with your fingers. The lysate was added according to the ratio of 150-250 microliters of lysate per cell of the 6-well plate. Then flick it with your finger to fully split 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.
3. After full cleavage, centrifuge 10,000-14000g for 3-5 minutes, take the supernatant, and then perform subsequent PAGE, Western and immunoprecipitation operations.
Description of the amount of lysate: Usually 150 microliters of lysate per well of cells in 6-well plates is sufficient, but if the cell density is very high, the amount of lysate can be appropriately increased to 200 microliters or 250 microliters.
In the experiment, different cells were cleaved in different ways. Such as grinding, cell homogenate, TritonX--100, dilute subtraction and so on. So, how do you choose the method of cell lysis? This section focuses on the detailed introduction of cell lysis methods in tissue samples.
1. Cut the tissue into tiny pieces.
2. Melt RIPA lysate and mix well. Take the appropriate amount of cracking solution, and add PMSF within a few minutes before use, so that the maximum final concentration of PMSF is 1mM.
3. Add the lysate at a ratio of 150-250 microliters per 20 mg 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.)
4. Homogenize with a glass homogenizer until fully cracked.
5. After full cleavage, centrifuge 10,000-14000g for 3-5 minutes, take the supernatant, and then perform subsequent PAGE, Western and immunoprecipitation operations.
6. If the tissue sample itself is very small, it can be properly cut and directly added to the cracking solution for cracking, and the sample can be fully decomposed through strong vortex. Then the supernatant was also centrifuged for subsequent experiments. The advantage of direct cracking is that it is more convenient, and it is not necessary to use a homogenizer, and the disadvantage is that it is more fully cracked than that using a homogenizer.
Note: It is normal for a small group of transparent glue to appear in the pyrolysis products of RIPA lysate. The transparent glue is a complex containing genomic DNA and so on. The supernatant can be directly centrifuged for subsequent experiments without detecting the protein which is closely bound to genomic DNA. If a protein that is particularly tightly bound to the genome needs to be detected, the transparent glue can be broken up by ultrasound treatment, and then the supernatant can be centrifuged for subsequent experiments. If some common transcription factors, such as NF-kappaB, p53, etc. are detected, these transcription factors can usually be detected without ultrasound treatment.
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