trouble getting cysteine labeled with biotin-maleimide - cysteine scanning purified protein (Aug/28/2006 )

Help! I cannot get my purified protein to label with biotin-maleimide! There is one cysteine, I inserted it, and have had it sequenced, so I know its there! I have found many protocols online, however none that fit in with my exact specs.

I am working on a membrane bound protein of the type three secretion system in Salmonella, but I am purifying it prior to any labeling

Here's my previous tries:

1. Purf protein (status checked and OK)- resuspend in PBS- the biotin-maleimide I use IS water soluble, BTW
2. % divide in half, boiled with 2% SDS for 10 min. (the other half of sample remains on ice)
3. Used reducing slurry and extract
4. 10mM biotin-PEO2 maleimide for 2 hours
5. Use a centricon column to bring down volume
6. Add loading buffers and run on SDS-page gel.

I talked to my committee, and first they said perhaps inclusion bodies were preventing the labeling of the denatured protein. So in the case, tried to denature with Guanidine-HCl for 15-20 min at 70Celsius. However, then you must precipitate protein out (with TCA) b/c Gu-HCl precipitates and will not run on SDS page gel. When I did this, I lost all protein. Seems like the biotin started to crystal?

Trying to develop a positive control which will include a cysteine in an accessible area of the protein monomer, however, this is a membrane protein and little bioinformatic work has been done on it.

Does anyone have a simple protocol to label a cysteine in a membrane-bound protein that has been purified? Or is there a way to get the excess biotin out before trying to precipitate protein with TCA?

Does 3K centricon column remove biotin-maleimide from the sample? Could I use the column, then precipitate out the protein, then resuspend that?

I'm so lost! I've tried many variations to the above protocol!

Anything inherently wrong with this protocol?

Thank you!

what is 3) reducing slurry?