Phosphorylated protein to do WB small coup
1, must add protease inhibitors in Lysis Buffer, but also add a certain amount of phosphatase inhibitor;
2, after adding the primary antibody, preferably 4 degrees overnight, to ensure that the antibody has sufficient binding time, because the phosphorylated protein only accounts for a very small part of the total protein;
3, choose high-quality phosphorylated antibodies, so to choose a good manufacturer, Novus is a global high-end antibody brand, its phosphorylated antibody works well;
4, the dilution factor of the antibody should be optimized, can be optimized according to the manufacturer's recommendations;
5. Dilute the antibody with 5% STSA-containing TBST, do not use the common 5%-Fat Milk TBST;
6. The background of phosphorylated protein WB is often deeper, so the tableting time should be appropriate, not too long or too short. If it is too long, the background will cover the strip that is too deep. If the time is too short, there may be no strip. The belt or strip is too shallow;
7. When phosphorylating protein WB, pay attention when washing with TBST. The rotation speed of the shaker should not be too fast. Do not wash the time too long. After incubating the primary antibody and the secondary antibody, wash it for 5 minutes and then 3 times. Some are better than not being able to do it. In addition, when washing, it is best not to stack a few films together.
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