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| Western Blot Forum Discuss western blotting and immunoblot in the western blot forum. |
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| My biggest tip for western is to make sure that your protein preparation is optimal, for mammalian cells I recommend CHAPS lysis buffer with incubation on ice. Also to know ahead of time if you want to reduce the proteins or run them in native state. |
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| sorry.. 1) Bubbles Sometimes bubbles are an issue. I use a roller for gels (biorad) that I use to roll over the nitrocellulose once put on the gel and then again on the whatman paper that goes over that, that makes sure that no bubbles are present. Also make sure that everything stays really wet and goes straight into the transfer tank (already filled with buffer) Drying out of the nitrocellulose can also result in bubbles and bad transfers. 2) Weak signals Can be the result of too little primary or secondary antibodies and this needs to be titered out. Usually it's a primary that can go to less dilutions and not so much the secondary, but each case can be different. 3) Static signals on the film These look like blobs everwhere. sometimes caused by static, to get a really nice looking blot, I take my probbed nitrocellulose, after putting ECL and place it in a plastic sheet protector. I then reduce the static by wiping down with a dryer sheet. And then finally put the film on to develop... removes static everytime! These are all I can think of off the top of my head. I've been doing westerns for many years though and have all sorts of tips if the memories can be triggered!! If you have an more specific problems, post them! |
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| Hi Molecule 2005, I really like your idea of a troubleshooting guide for Western Blot. We have problems with blotchy Western Blots. It looks like some detergent / fat a.s.f. at some point is getting to the membrane. We use semidry blotting, antibodies in milk, blocking in milk 3hrs RT, blocking & secondary ab in weighing plastic trays, hybridization primary ab in 50ml falcons. Any suggestions would be greatly appreciated! Thanks, Daniel Last edited by damertens1; 02-25-2008 at 12:28 PM. Reason: link doesnt work |
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