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-   -   Troubleshooting help, please! (http://www.molecularstation.com/forum/western-blot-forum/72934-troubleshooting-help-please.html)

Vaappen 05-08-2010 05:28 PM

Troubleshooting help, please!
 
Hi everyone,

I'm having some troubles with my Western blotting. I'm using nitrocellulose membranes and 40ug of protein per lane. When staining the filter with Ponceau S, I can detect strong protein bands, but after the antibody incubations I can not detect anything on the film - not even with 1:1000 diluted Rabbit anti B-tubulin (1h at room temperature) and HRP Goat anti Rabbit (1h at room temperature) antibodies. I use Nacho's blocking solution (1h at room temperature) and Thermo Scientific West Femto chemiluminescent substrate solution.

What could be the problem?

With PVDF membranes I had a strong signal with the same protein lysates, so there must be something wrong with the blotting or blocking the membrane or with the antibodies.

Eika 05-12-2010 09:26 PM

Re: Troubleshooting help, please!
 
hi
Try to incubate your anti beta-tubulin antybody at 4C overnight.

Bonnie 05-13-2010 05:00 PM

Re: Troubleshooting help, please!
 
If I were you I'd just switch back to PVDF membranes. :D Don't go changing something that's working.....


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