quantifying "ghost" bands - (Jun/15/2010 )
I read about the white bands (normally called ghost) here in the forum. I had those bands more than once in my control protein, beta-actin, probably due to an excess of primary antibody (I used a 1:250 dilution in the first time and 1:1000 in the second). Although those bands appear white I can still quantify them using Image J but I don’t know if this procedure is correct. So my question is: may I use those results or not?
I also tried to strip those membranes but after the stripping I incubate them with the secondary antibody to verify if the primary had been all removed. Instead of obtaining a “clean” film, as I expected, I found that the beta-actin bands were still there, although this time they appeared with the regular dark shape. Once again I have doubts if I should quantify these bands because I don’t know if the stripping removed the antibody in excess equally in all samples.
Any suggestions to solve this issue?
You can dilute your antibody even further. My beta-actin antibody I use 1:50,000 (Yes, one ul per 50mL) and it works fine. If you take the membrane and the ECL into the darkroom, turn off the lights and dump the ECL on the membrane you will actually see the blue light coming off the membrane. What is happening is that by the time you get to the darkroom and set film, the HRP has used all the available substrate and hence, you are getting the ghost bands. Quantifying bands from a stripped membrane seems to be a topic of debate as some people will say it's ok while others will say it's invalid. Personally, I think it's ok. The entire membrane was stripped so the removal of antibody should be equal across all lanes. But...I'd also say you should do it again just to be safe. You can not quantify the ghost (white) bands.