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Position: Discussion forums > Bioscience biotechniques discussion forums > Proteomics and protein biochemistry forum
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No band at film developing ( Western blot)
Posted by: minsu (IP Hidden, New member, 1)
Date: August 11, 2005 05:57PM
Hi!
I am testing with the same antibody. At the very first time, it formed bands at the film. But at the next times (I tried 4 times) there is no band at all. All are with the same procedures, including the amount of antibody. Could you explain me the reason why and how can I solve it. Thanks in advance, Best regards, Minsu.
Re: No band at film developing ( Western blot)
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: August 12, 2005 03:05PM
There are many things that may have happened. In your repeated attempts are you using the same membranes? Or have you transferred sample to a second membrane? Several things may have happened: (1) primary or secondary antibody not properly stored at -70C (2) antibody has lost effectiveness due to repeated freeze/thaw cycles, (3) the proteins were stripped from the blot by a too strenous protocol, (4) there was a poor transfer in subsquent attempts, (5) less protein was loaded onto the gels in subsquent attempts. If you are restaining the same membranes, I think repeated stripping of the antibody may have removed some of your target. This will be more difficult for the antibody to detect. Try increasing your incubation times or extending the amount of time the blot is exposed to the film to compensate for the lost target.
Re: No band at film developing ( Western blot)
Posted by: Chenoeh (IP Hidden, New member, 5)
Date: August 18, 2005 09:55AM
You could also stain the membrane in Ponceau S after transferring and before blotting to check that the proteins have transferred properly.
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