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black border on blots
I consistently have a black border around the edges of my membrane that shows up on film. Sometimes it is minimal, other times it is quite blaring. I can often see it in the darkroom with the naked eye. When it is blaring, it billows inward and sometimes interferes with my ability to quantify bands. Sometimes, some "cloudiness" also billows inward from the black border. I use Pierce Super Signal, and 1% fish gelatin for blocking. I have tried adjusting secondary antibody, filtering gelatin and diluted antibodies. I have added many washes with TBST, TBS and water at various stages. The black border is always there, just in varying degrees, with no pattern relating to the things I have tried. I have attached an image of an example of a more blaring border. It is a 30sec. film exposure. I also use a film cassette that has a white glossy interior. Could that be causing the problem?
Any suggestions would be appreciated. Thank you.
Re: black border on blots
I've had this happen to before. Simply, reduce the amount of ECL (Pierce Super Signal). I am curious to know how much ECL in mLs you are using? Good Luck.
|black , blots , border|
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