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Tryptic digest help - Trouble diffusing stain (Jul/24/2006 )

Hi all,

I am wanting to do a tryptic digest on spots obtained through 2D gel electrophoresis. I excised the spots from the gel and placed in acetonitrile, however, it is taking ages for the stain (Biorad Biosafe Coomassie) to diffuse out of the gel. Has been a few days - changing the acetonitrile each morning to keep it fresh.

Any ideas why this is taking so long? Any suggestions or advice would be much appreciated.

Thanks

-btjd-

QUOTE (btjd @ Jul 24 2006, 09:52 PM)
Hi all,

I am wanting to do a tryptic digest on spots obtained through 2D gel electrophoresis. I excised the spots from the gel and placed in acetonitrile, however, it is taking ages for the stain (Biorad Biosafe Coomassie) to diffuse out of the gel. Has been a few days - changing the acetonitrile each morning to keep it fresh.

Any ideas why this is taking so long? Any suggestions or advice would be much appreciated.

Thanks


is the spot being shaken as this can help, unfortunately coomassie is quite a permanent stain you could try an alternative destain (10% acetic acid, 5% MeOH)

you may want to try an alternative stain i have used 0.2M Copper chloride which negatively stains the gel, the destaining with EDTA (sorry cant remember what conc.) is almost instantaneous.

much better is that sensitiviyt is simehwre between coomassie and silver!!!

-Jimmy_september-

hello,

you can try using larger volumes than what you're currently using for de-stain. also, you can chop your gel pieces into smaller fragments to increase your surface area to volume ratio. don't worry too much if your piece isn't completely de-stained. i've been able to get good spectra on spots that had faint stain. just be sure to clean up your digested peptides before MS analysis w/ a zip tip or some other reverse phase media. if you're doing LC-MS/MS, i'm pretty sure the on-column wash will be sufficient to remove the coomassie.



QUOTE (btjd @ Jul 24 2006, 08:52 PM)
Hi all,

I am wanting to do a tryptic digest on spots obtained through 2D gel electrophoresis. I excised the spots from the gel and placed in acetonitrile, however, it is taking ages for the stain (Biorad Biosafe Coomassie) to diffuse out of the gel. Has been a few days - changing the acetonitrile each morning to keep it fresh.

Any ideas why this is taking so long? Any suggestions or advice would be much appreciated.

Thanks

-johanski-

Destain in 200 mM Ammonium bicarbonate and 40% acetonitrile at 37 degrees and it will destain within half an hour. If not completely destained refresh the solution after half an hour


QUOTE (johanski @ Aug 2 2006, 11:23 AM)
hello,

you can try using larger volumes than what you're currently using for de-stain. also, you can chop your gel pieces into smaller fragments to increase your surface area to volume ratio. don't worry too much if your piece isn't completely de-stained. i've been able to get good spectra on spots that had faint stain. just be sure to clean up your digested peptides before MS analysis w/ a zip tip or some other reverse phase media. if you're doing LC-MS/MS, i'm pretty sure the on-column wash will be sufficient to remove the coomassie.



QUOTE (btjd @ Jul 24 2006, 08:52 PM)

Hi all,

I am wanting to do a tryptic digest on spots obtained through 2D gel electrophoresis. I excised the spots from the gel and placed in acetonitrile, however, it is taking ages for the stain (Biorad Biosafe Coomassie) to diffuse out of the gel. Has been a few days - changing the acetonitrile each morning to keep it fresh.

Any ideas why this is taking so long? Any suggestions or advice would be much appreciated.

Thanks

-A.Snijders-