The purified protein only has one band in both SDS and after transfer in ponceu, But when DAB developer is used the entire lane of the blot is getting dark with multiple bands.
we are using nitrocellulose membrane for transfer. can someone suggest possible troubleshooting areas? Thank you.
Hello Ajieth Kanna Ss
Preparation of DAB solution for use in Western Blot.
Dissolve 50mg DAB in 100ml Tris Buffered Saline (TBS). Vortex the solution until DAB completely dissolves. Add 10 µl 30% H2O2. Make this solution just before use.
TBS contains (20 mM Tris, 500 mM NaCl, pH 7.5).
Immerse the nitrocellulose membrane in DAB color development solution. Proteins will become visible immediately as brown bands. Proteins of lesser concentration may take as long as 10-15 minutes. Do not overdevelop, as high background may result.
You may stop the color development by immersing the membrane in distilled water for 10 minutes. Change the water at least once to remove the remaining color development solution.
Do you follow a similar protocol as above?
The only disadvantage of using DAB is that it reacts slowly with H2O2, even without the presence of a peroxidase enzyme. So, whenever you combine DAB with H2O2, you will have to use the solution relatively quickly to avoid generating brown precipitate in solution, which is likely to contribute to background staining.
Best.
Hello Malcolm Nobre Thank you for your response.
I will try what you have suggested.
And I also found that the nitrocellulose membrane we are using is for chemiluminescence.
we do not have any facilities for doing xray or fluorescence imaging, therefore we can only develop with DAB.
Could this also lead to the problem? Please do let me know.
Thank you.
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