After developing a western blot recently, I noticed that one of the bands present contained white areas where the blot was not exposed. I’ve had this problem in the past, but it only seemed to occur after I had exposed the membrane to X-ray film for too long resulting in overexposure and very dark bands. This bubbling issue would occur upon repeated attempts to develop the film. In this case however, I properly calibrated the antibody concentrations based on previous attempts, and this was the result after only one attempt of development. Both bands are of appropriate intensity, but the lower band does not come out well. Any suggestions?
Hi, there are no bubbles. What you see is bleaching, due to too strong signals on the blot (too much protein / too high concentration loaded, too low dilution for first and/or second antibody etc.).
To avoid this, I would propose to generate a higher dilution for the sec. antibody (1:5,000-1:20,000). In the next step maybe use higher dilution factors for the primary ab and/or reduce the concentration of protein in the loaded sample (by higher dilution).
Hi,
The problem might be due the high concentration of protein (loaded sample) as well as the higher concentration of antibody.
Andreas Eisenreich and Mohammad Nasiruddin are right. Its bleaching. Too much protein under high antibody titres cause this issue.
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