mass spec with silver stain band, unsecssessful. help!1 - (May/16/2007 )
I 've been tried to identify my protein complex by using mass spec with my silver stain bands. I see very strong and clear band on my silver stain gel, but no secess so far. Anyone use silver stain bands mass spec sucessfullly? and where did you get that done? I suspect that the facility is not good enough. I am in UK now. Any tips will be helpful. Thanks a lot
Cathy
Hi Cathy,
I'm about to do the same thing... anyway, the protein analysis facility in our institute recommends colloidal blue staining (Invitrogen?) instead of silver staining
(which could cause some troubles, because you have to destain and so on) for mass spec.
Cathy
it really depends on facility and equipment; as suggested, try to stain with colloid CBB (f.i. of Pierce); if you have clear silver stain, you also may succeed with CBB, or enrich your protein of interest
hi cathy
if you are using a classical silver staining method there's no way for you to use mass spec.
there exist a protocol for silver staining compatible with mass spectrometry (if you search it's not difficult to find), but you must re-prepare the gel from the beginning.
Furthermore silver staining is sensitive enough to let you visualize protein in extremely low quantity, often not sufficient to be identified.
if you are using a classical silver staining method there's no way for you to use mass spec.
there exist a protocol for silver staining compatible with mass spectrometry (if you search it's not difficult to find), but you must re-prepare the gel from the beginning.
Furthermore silver staining is sensitive enough to let you visualize protein in extremely low quantity, often not sufficient to be identified.
Thank you all for the suggestion!!. I decide to use silver stain. the reason is that after two step purification, I was left with very little protein. the starting amount is already 1700ml culture. If I want to see it on Coomassie, I guess minimum i need to use 20times more which is a hugh number of cells, taking the whole incubator!!!!!
And I do not know is it going to work?
Gosh.
if you are using a classical silver staining method there's no way for you to use mass spec.
there exist a protocol for silver staining compatible with mass spectrometry (if you search it's not difficult to find), but you must re-prepare the gel from the beginning.
Furthermore silver staining is sensitive enough to let you visualize protein in extremely low quantity, often not sufficient to be identified.
Thank you all for the suggestion!!. I decide to use silver stain. the reason is that after two step purification, I was left with very little protein. the starting amount is already 1700ml culture. If I want to see it on Coomassie, I guess minimum i need to use 20times more which is a hugh number of cells, taking the whole incubator!!!!!
And I do not know is it going to work?
Gosh.
another alternative is staining with fluorecence protein staines which detect in the nmolar range as silver; it should not interefere with Ms/Ms but ask the administrator of the facility...
if you are using a classical silver staining method there's no way for you to use mass spec.
there exist a protocol for silver staining compatible with mass spectrometry (if you search it's not difficult to find), but you must re-prepare the gel from the beginning.
Furthermore silver staining is sensitive enough to let you visualize protein in extremely low quantity, often not sufficient to be identified.
Thank you all for the suggestion!!. I decide to use silver stain. the reason is that after two step purification, I was left with very little protein. the starting amount is already 1700ml culture. If I want to see it on Coomassie, I guess minimum i need to use 20times more which is a hugh number of cells, taking the whole incubator!!!!!
And I do not know is it going to work?
Gosh.
I have carried out the experiment like that, at first, silver staining is processed in order to improve the sensitivity of detection, but when u are about to prepare the sample for mass spec, CCB is better. if u can not detect the band with CCB, silver staining is OK, what u must keep in mind is that the sample for mass spec needs desilverization after silver staining
I have a good silver stain protocol, modified from that of Schevchenko, that is completely compatible with MALDI. The other alternative is to stain with Sypro Ruby, then counterstain with Coomassie G250 (will give you sensitivity down to about 1ng).
I've had good results with both.