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Technical Column: Precautions for Phosphorylated Protein WB Experiment
Protein phosphorylation is a very important post-translational processing, and detecting protein phosphorylation is necessary for signaling pathways. Western blot is the most commonly used method to evaluate protein phosphorylation status, which is not much different from running normal proteins, but incubating antibodies that recognize phosphorylation. Below, Hengyuan Biotech will summarize the key points to pay attention to in the experiment.
1、 Regarding phosphorylated protein samples
The phosphorylation of protein in lysate is a very rapid reaction, and the sampling process should be fast. The sample should be fresh, and it is best to operate on ice for sample processing, with the operation time as short as possible. When washing cells with PBS, PBS must be pre cooled at 4 ℃. If it is an organization, liquid nitrogen grinding method should be used for protein extraction, and the grinding tool should be pre cooled in advance to maintain a low temperature state.
It is best to prepare a fresh lysis solution for extracting phosphorylated proteins. The lysis solution must contain protease and phosphatase inhibitors, otherwise even if the bands are pressed out, the results will be shallow and unreliable. Okadaic acid, NaF is a phosphatase inhibitor. Furthermore, it also depends on the amino acid phosphorylation site of the protein you test. If it is tyrosine, 1 u M of sodium vanadate, also known as sodium orthovanadate, should be added. Do not boil before loading the sample, as boiling may damage its phosphorylation site.
2、 On the issue of background and stripes in WB
When phosphorylating protein WB, the background is often deep, so the compression time should be appropriate, not too long or too short. If it is too long, the background is too deep to cover the desired band. If the time is too short, there may be no band or the band may be too shallow. When performing phosphorylated protein WB, non-specific bands are often present in addition to the target protein bands. If the phosphorylation antibody is not good, it may even produce non-specific bands, but there may not be the band you want. Therefore, after pressing the tablet, it is necessary to compare the molecular weight of the bands you have pressed with Markers to see if it is correct.
3、 On the issue of total protein expression level
After studying the phosphorylation of a certain protein, it is best to also investigate the total expression level of that protein. There are two methods for this. One is to load the same sample twice in different wells (either on the same gel or on different gels), with one pressing the phosphorylated protein and the other pressing the total expression level of the protein (including phosphorylated and unphosphorylated proteins), and even running another gel to press the internal standard. However, it is generally not recommended to do so because there is still a significant margin of error when comparing. Therefore, it is widely recognized and commonly used method to wash off the previously bound phosphorylated antibodies and secondary antibodies with strip solution, and then use the same membrane to compress the total protein. Then elute again and press the internal standard again.
For more experimental information, please consult our sales representative. Shanghai Hengyuan Biotechnology Co., Ltd. has its own laboratory and a professional technical research and development team. It has been committed to the research and sales of various biological reagents, cells, serum, and ELISA kits for a long time. We provide free testing services for experiments and exquisite technical services. If you have any questions about experiments, please feel free to come and consult us, and we will provide you with professional one-on-one technical guidance.
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