http://www.ludwig.edu.au/www/jpsl/JPMETH.html
I thought you might like to look at this method and compare to what you're
doing now. A brief glance tends to indicate you may washing too thoroughly!
Hope this helps,
Roger
At 11:56 AM 14-07-98 +0000, you wrote:
>Dear ABRFer's,
>
>Recently our lab has started protein and gel work. We have had
>trouble digesting the proteins from an acrylamide gel matrix and
>hope that some of you have suggestions. We are using apomyoglobin as
>the trial protein, modified sequencing grade trypsin from Boehringer,
>and 8% polyacrylamide gel under denaturing condition. We typically
>stain with Coomassie Blue (0.1%) for 30min and destain with 40% MeOH,
>10% acetic acid overnight with several changes of solution. After
>destaining the bands are excised, washed several times with ammonium
>bicarbonate and ACN to release the Coomassie stain. The gel pieces
>are dried, disulfide bonds reduced with DTT, and treated with
>iodoacetamide to prevent reformation of disulfide bonds. After
>rinsing and drying the gel is swelled with the digestion buffer and
>incubated overnight at 37C. Recovery is performed using 0.1%TFA and
>ACN. We are trying to detect the peptides using a C18 narrow bore
>column for which we have a method which works well with proteins
>digested outside a gel. We think that we are losing the proteins at
>one of these steps but have been unable to determine which so far.
>Any suggestions would be greatly appreciated. Thanks,
>
>Dan
>
Roger Murphy, Ph.D.
Biological Production Facility
Ludwig Institute for Cancer Research
Austin & Repatriation Medical Centre
Studley Road,
Heidelberg, Vic. 3084
Australia.
Tel 61-3-94965463
Fax 61-3-94965436
Email murphy_r@licre.ludwig.edu.au