First, because the phosphorylation of proteins is reversible and will be dephosphorylated by phosphatases, it is necessary to inhibit or avoid the interference of endogenous and exogenous phosphatases in the sample preparation and the entire immunoassay process. For the endogenous phosphatase of the cell, we need to add a sufficient amount of fresh phosphatase inhibitor to the cell lysate during sample preparation, such as PhosphoSafe series. Exogenous phosphatase is mainly brought in by other reagents during the experiment. For example, we must ensure that the blocking agent does not contain phosphatase. At the same time, the bacteria contained in the sample or other reagents will also secrete exogenous phosphatase, so another key to phosphorylation of Western Blot is to use clean fresh solution and newly made SDS polyacrylamide gel , And it is best to load the sample immediately after the sample preparation, and complete the SDS-PAGE and Western Blot detection on the same day. If you want to use the same Blot for whole protein immunodetection, you need to do the phosphorylation test first and then the whole protein test to avoid the loss of phosphorylation signal. This is very important.
Some experiments use Western Blot to verify the presence of a group of phosphorylated peptides or proteins. At this time, a universal phosphorylated antibody is needed. Because universal phosphorylated antibodies, such as 4G10 tyrosine phosphorylated antibodies, have a wide recognition range and good phosphorylation specificity, they will not cause false negative results.
Taking Liu's research as an example, they used the universal anti-tyrosine phosphorylated antibodies 4G10 and PY20, using Western Blot technology to verify the results of the peptides screened by the peptide chip. Their laboratory was designed in this way. First, a phospho-tyrosine peptide array was used to screen the phosphorylated peptide sequences bound to the SH2 domain, and then Western Blot verification of these synthesized peptide sequences , The phosphorylation levels were detected using the universal anti-tyrosine phosphorylated antibodies 4G10 and PY20. They found that different antibodies have very different recognition specificities. For example, pTyr-Pro sequences can be bound by certain SH2 domains, but not by 4G10 and PY20. But in general, the universal anti-tyrosine phosphorylated antibodies can recognize most of the phosphorylated polypeptides bound by the SH2 domain, especially the 4G10 antibody can recognize more phosphorylated tyrosine polypeptides than the PY20 antibody.
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