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Protein Transfer Problems from SDS-PAGE Gels - Involves Coomassie Staining (Apr/07/2010 )

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For ponceau, I let act the solution 5 minutes on my membrane, it is enough. I wash then 3 to 4 time with large volume of TBS-T (until my washing buffer stop to be pink).

To my mind, two things to do:

1 Try your ponceau and coomasie solution on old membrane already detected by colleague. If the staining is not working, try other recipies.

2 When this staining is working, make a new blot, keep two membranes in your transfer protocol. Try to expose the membrane in contact with the gel with ponceau, and the other with Coomasie and labbel the gel with Coomasie.

Ps. Stupid question, but are you blotting in the good sens? gel on the - and membranes on the + ?

-nicolas-

Yup, I am blotting in the correct sequence- I mean unless BioRad suddenly decided to make black positive and red negative haha.

I suppose the reason I am have some trouble with the troubleshooting is because no one in my laboratory has done western blotting before. I am trying to build the protocol from the ground up.

I think next time I run the blots, I'll stick to the new buffer concentration and put membranes on both sides of the gel, just in case.

-Chiapet874-

Bah, still no bands on transfer membranes. =(

Is there a polarity to these membranes? Also, when I take the transfer membranes out of the cassette post-transfer, I see "grayish" areas, does that mean there were air bubbles there?

-Chiapet874-

0.1% sds in the transfer medium is too much. try 0.05%. keep the methanol at 20%.

the methanol strips sds off the protein. sds interferes with binding.

0.05% sds helps get the protein out of the gel and then gets removed by the methanol.

-mdfenko-

Hmm, I will try that out!

I have a couple of transferring troubleshooting ideas up my sleeve- but I was wondering, can I use the Biosafe coomassie stain to blot my membranes for proteins? I hear that coomassie stains are more sensitive than Ponceau stains, so I suspect that maybe I will get different results with a different stain.

-Chiapet874-

Chiapet874 on Apr 18 2010, 09:49 PM said:

I have a couple of transferring troubleshooting ideas up my sleeve- but I was wondering, can I use the Biosafe coomassie stain to blot my membranes for proteins? I hear that coomassie stains are more sensitive than Ponceau stains, so I suspect that maybe I will get different results with a different stain.

in general, no. coomassie stain is permanent. ponceau s is easily removable. coomassie will interfere with color deposition (used in some western blots) and may interfere with chemiluminescence. it may also interfere with antibody binding.

since you are using chemiluminescence to visualize your blot you can try it, if you don't mind possibly wasting the materials.

-mdfenko-
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