Dear all,
I transferred proteins labeled with a fluorescent dye from (a part of) a 2D SDS-PAGE gel to a nitrocellulose membrane. After scanning the nitrocellulose membrane to check whether the protein of interest was transferred (i.e., before antibody incubation), I observed huge (and smaller) black dots. It's certainly not a protein spot. I was wondering what causes these black dots. Does someone experienced the same problem or know what causes these black dots?
(The membrane isn't damaged, I can't see dots with the naked eye, glass plate where the membrane was put on for scanning was clean, the membrane was dry during scanning.)
Thanks in advance.
Kind regards,
Valérie
It looks like something got spattered onto the membrane. I suggest starting again with a fresh membrane that you are sure hasn't been contaminated.
Hi Valérie, what is the fluorescent dye you are using to label proteins? If it close to UV then any and all handling of the membrane will give background spots.
Hi Adam and Steingrimur,
I'm always using fresh membranes of which I'm 100% sure they are not contaminated. So I'm quite sure contamination is not the problem.
I am using T-rex labels (company: dyeagnostics) with the following specifications:
Exitation max: 650 nm
Emisssion max: 665 nm
Filter settings: G-Dye300, Cy5, Alexa 647
Thanks for your interest in my question and your suggestions.
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