I am trying to detect the phosphorylated IKK protein using Western Blot. I induced the cells by 10ug/ml LPS for 20 min. I loaded approximately 25 μg of total lysate and ran the gel. After gel transfer, I blocked the membrane using 5% BSA in TBST (0.1% Tween-20) for 2 hours. Then, I incubated the membrane with a primary antibody (diluted 1:1000 in blocking buffer as recommended by the manufacturer) at 4 degrees overnight. Next, I washed the membrane three times for 5 minutes each with TBST. Following that, I added a secondary antibody (diluted 1:2000) and incubated for 1 hour at room temperature. Finally, I washed the membrane three times for 5 minutes each with TBST before proceeding with chemiluminescent visualization.
However, I have been encountering a problem where I observe numerous nonspecific bands, and my desired band appears to be very faint. I attempted to address this issue by blocking the membrane for a longer duration using 7% BSA, but it did not make any difference. Have you encountered a similar problem with the detection of phosphorylated proteins, and if so, what solutions would you recommend? Thank you!
Hello Quynh Pham
The protocol seems to be okay except for some modifications.
1. Try optimizing the LPS concentration and the time point for induction.
2. Try to reduce the concentration of secondary antibody. Use of too high concentration of secondary antibody may result in non-specific binding to proteins other than the protein of interest. Non-specific binding can occur with any protein. Thus bands of higher and lower molecular weight may be observed. Secondary antibody in the range of 1:15000 -1:20000 could be used. It’s a good practice to titrate antibody concentrations, thereby optimizing the signal to noise ratio. You may run secondary antibody control without primary to confirm specific binding of primary antibody.
3. Has the sample been completely reduced? Incomplete reduction of sample can lead to bands containing high order species which appear as bands at unexpected sizes or as smears. Use fresh reducing agent such as BME or DTT in sample loading buffer and boil in SDS (5-10 minutes).
4. The possibility of sample contamination may be another factor which is less likely to happen. Bacterial growth in running buffers can lead to unexpected bands on the gel. Always handle gels wearing gloves and use fresh buffers each time.
Hope these may help!
Best.
@Malcolm Nobre thank you for your suggestion! I fixed this problem by reducing the concentration of the second antibody and changing the blocking buffer from BSA to non-fat milk!
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