I am doing Ubiquitination assay of my target protein. Unluckily, most of the time i got black background hiding the smeared ubiquitinated protein. i tested it by changing various experimental parameters, (like different % of non-fat skimmed milk, washing, antibodies conc. and time etc) but still unable to reduce the background shade. Kindly share your experience.
Thanks
Hi Shah, based on the figure you show, it looks like you are using luminescence to expose film, right?
To me, it looks like that the cassette that you are using to illuminate the film is leaky, meaning it lets in light.
If this was a background problem then you would expect to see non-specific bands showing up. But your picture shows blacked-out regions of the whole gel that do not correspond to non-specific binding.
If this is film exposure, then wrap the cassette in black plastic after placing the blot with the film in the darkroom.
Thank you Stefansson, yes i am using luminescence. i will consider your suggestion. But i get these dark shades only in using Ubi Ab. in case of using other primary antibodies i had a clear background.
Regards.
shah
Hi, if you only get these blacked out sections using the Ubi Ab, then there is something very wrong about the Ab itself.
Non-specific background binding is common problem in luminescence. But to have whole sections of the film blacked out is something that I have not seen before, barring a leaky cassette.
How long do you expose the film to the blot? If it takes more than 1 min to see anything, then the signal is too weak.
Try another Ubi Ab from a different vendor.
Hi Stefansson.
The exposure time is 5 min, your point is valid regarding Ab. I order the Ab about a year ago. Let me try using the fresh Ab.
Thank You for your kind guidance.
Regards
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