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Position: Discussion forums > Bioscience biotechniques discussion forums > Proteomics and protein biochemistry forum
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Western blotting
Posted by: shoba amarnath (IP Hidden, Unregistered user, )
Date: April 17, 2005 09:32AM
Hi,
I have performed a western blotting and the transfer has worked and i could see bands on my PVDF membrane using visualization dye. The problem is when i used actin antibody, i had no bands on the film when developed. also i know the antibody as worked since i have made it to work before. The secondary used is also correct. Can anyone give me any suggestions why?? Ineed these results for a paper so iam in a fix.
Re: Western blotting
Posted by: bassamfahmawi (IP Hidden, Regular member, 42)
Date: April 18, 2005 10:26AM
Hello,
I am not familiar with the moleucalr weight of Actin, what is the molecular weight? At what voltage or mA do you transfer your blot and for how long? do you use semidry or a liquid electroblotting? Providing these infromation will help better trouble shoot your problem.
Re: Western blotting
Posted by: Heatherbee (IP Hidden, New member, 3)
Date: April 19, 2005 11:01AM
I've used actin before, and it is unusual that it didn't work. If you have blocked the blot for too long, sometimes bacterial contamination can interfere with the primary binding. I know for a fact that blots can be stripped in a buffer containing beta-mercaptoethanol for 30 minutes @ 50 degrees C and blocked and reincubated in the primary for actin. Actin is a very strong reacting antibody usually. Perhaps the antibody is old or contaminated?? If it is it will fail, sometimes all of a sudden.
Unfortunately, the thing with westerns is occasionally they don't work for any good discernable reason....I'd suggest stripping and redoing the blot. Or start from scratch with a fresh gel if you have sample to spare.
Re: Western blotting
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: April 20, 2005 01:55PM
Hi,
I have also used actin before. We strip our blots using the same process as heatherbee and still find a strong actin signal. Are you using the same dilution of primary + secondard antibodies as before? Has your source of actin changed? If so the required dilution factor may have changed. Does a band of the same apparent molecular weight as actin appear on the PVDF membrane when visualized by dye? If not then it is possible that actin did not transfer. If this is the case vary your transfer conditions. Perhaps add SDS to the buffer or transfer for a longer period of time at a lower voltage.
Re: Western blotting
Posted by: bassamfahmawi (IP Hidden, Regular member, 42)
Date: April 20, 2005 02:06PM
A gentle way to strip your blot without the use of the smelly ME, is to strip your blot with 0.2 M glycine pH 2.5 at room temperature for 15 minutes and if you do not get complete striping you can extend the incubation of your blot. Try it and you well smell the difference! Good Luck!
Re: Western blotting
Posted by: femmeauburn (IP Hidden, Advanced member, 115)
Date: April 22, 2005 09:23AM
You may wish to add 0.05% Tween-20 to the 0.2M glycine pH 2.5 stripping buffer.
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