I have a western blot that I am visualizing with a colorimetric solution and I have no bands. I know there is something on the membrane however, because I stained it after transfer. I blocked in skim, applied primary 12-24 hours, applied secondary 75 minutes, and visualized. On the first trial secondary stayed on quite a bit longer, and I had no result. Nothing at all showed up on the membrane. The second trial, I did the secondary for exactly 75 minutes, visualized, and nothing showed EXCEPT my ladder. So, now I know the secondary and visualization worked, but maybe not the primary? I have never had issues with this primary before, so I am not sure. Aside from there simply not being any of the specific protein on there, I am trying to look at other angles first. I can attach my pictures of membranes after visualizing, the first trial is the cloudy/bubbly looking one, the second is the one with the ladder.
Hi Abby, could it be possible that the protein isn't running through the gel or transferring onto the membrane? I'd recommend using Ponceau staining to verify bands are present before continuing with primary/secondary.
I know things ran and transferred because I have been staining each step of the way. So I stained my gel with Gel Code Blue and got a picture to verify proteins. Then when i transfered the Gel Code Blue also transferred, so i kkow it got onto the membrane. I quickly snapped a picture with my phone before it washed away with the block but I know there’s something on there.
Dear Abby,
I think you have a problem with your secondary antibody or with the blocking buffer and mostly the last one. Make a fresh blocking buffer and incubate with for one hour, if you always wash after blocking, do not wash pls and vice versa.
To make sure about your primary antibody, you can test them in immunofluorescence.
Could your problem be with the color metric solution? Are you using HRP conjugated ABs? Is your colorization agent DAB? If so, check the age of it. They do go bad over time and it's best to store them in the freezer when not using them.
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