Protocol of Western blot for Phosphorylated Proteins
Licia Miller Product Manager
Phosphoprotein western blot is a widely used technique for detecting and analyzing the phosphorylation status of proteins, which is essential for understanding various biological processes and signal transduction pathways. Some common application scenarios include signal transduction research, cell cycle and proliferation research, disease mechanism exploration, drug development, neuroscience research, biomarker discovery, etc.
When performing western blot analysis of phosphorylated proteins, it is important to use specific antibodies that recognize the phosphorylated form of the target protein. In addition, optimization of sample preparation, gel electrophoresis, transfer efficiency, and detection methods are essential to obtain accurate and reliable results.
What is known is that when the target cells or tissues are homogenized in a freshly prepared lysis buffer, the lysis buffer used should contain a protease inhibitor cocktail, and similarly, when dealing with phosphorylated proteins, a phosphatase inhibitor cocktail should be included, for example, using RIPA or NP40 buffer supplemented with fresh protease and phosphatase inhibitor cocktails.
Second, it is important to note that once cells or tissues are lysed, proteolysis, dephosphorylation, and denaturation begin. These processes can be greatly slowed down by keeping samples on ice or at 4°C at all times and by adding fresh, targeted inhibitors to the lysis buffer.
Experimental Steps
1. Add an equal volume of 2× SDS-PAGE sample buffer to a protein solution sample containing 1-100 ng of target protein (500 µg of lysate) .
Note: For reduced samples, the sample buffer should be supplemented with DTT or β-mercaptoethanol.
For non-reduced samples, no DTT or β-mercaptoethanol was added.
2. Heat the sample to 95°C or boil for 5 minutes to denature the protein.
3. Load the samples onto SDS-polyacrylamide gel and perform gel electrophoresis under standard conditions.
4. Transfer the protein to PVDF membrane using semi-dry or wet transfer method.
Note: For PVDF, the membrane must be pre-wet with methanol prior to transfer.
5. If needed, transfer efficiency can be determined by briefly staining the membrane with Ponceau red dye (10 seconds) .
Note: To prevent the removal of proteins during the washing process, it is recommended not to wash the membrane with distilled water . Instead, use PBST or TBST buffer for washing.
6. Add 5% w/v BSA to TBST, and after it is fully dissolved, pour it into the container containing the membrane, place it on a shaker, and incubate it at 4°C for 1 hour to block the non-specific proteins on the membrane.
Note: The blocking solution should completely soak the membrane.
7. Dilute the primary antibody in TBST buffer according to the recommended dilution of the primary antibody product. Then pour it onto the membrane for primary antibody incubation and incubate overnight at 4°C with stirring.
Wash the blot three to four times with TBST at room temperature for 5 minutes each time.
9. Dilute the horseradish peroxidase (HRP) -labeled secondary antibody with TBST according to the recommended concentration of the secondary antibody product, then incubate with the membrane and stir at room temperature for 1 h.
10. Wash the membrane three to four times with TBST at room temperature for 5 minutes each time.
11. Detect the target protein according to the instruction manual of the ECL detection kit.
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