how can I get enough proteins for Mass spec? drive me mad! - (Aug/06/2007 )
After start material of 2 litre cell cultre, followed by two step purification, I see clear band on silver stain. But seems nobody could use silver stain band to get Mass spec working on it. So I tried to start with larger material to start, to get commassie stain level protein band for Mass spec. But it seems mission impossible 
. need really huge amount, gosh. drive me crazy recently.
anyone has any suggestion? Thanks a lot. save me from the hell!!!!!!!
1. Have you tried colloidal Coomassie ("blue silver")? (PM me to get a protocol)
2. If you have enough material you can try to run two lanes, cut gel after run and silver stain just one then match stained with not stained.
2. If you have enough material you can try to run two lanes, cut gel after run and silver stain just one then match stained with not stained.
1. I am using collodal coomassie.
2. i could do it without staining with silver, but the most important thing is that the protein con is still silver level, too low for MAss spec
You don't have antibodies for your target proteins, do you?
hi cathy,
I undestand you, 
I had a similar problem some months ago (I wanted to identify an inmunoprecipitated protein) and still no solution due to the high amount of IgGs. It seems impossible. 
One guy from other lab told me that he sent to the mass spec service several bands, from different gels, of his silver stained protein. He was not able to concentrate enough to see it in one coomassie stained gel, but he saw the band with silver. So he repeated that and cut off several (7-8) silver stained bands and the protein was identified.
Perhaps people from the mass spec service may help you in some way.
Good luck.
I undestand you,
I had a similar problem some months ago (I wanted to identify an inmunoprecipitated protein) and still no solution due to the high amount of IgGs. It seems impossible. One guy from other lab told me that he sent to the mass spec service several bands, from different gels, of his silver stained protein. He was not able to concentrate enough to see it in one coomassie stained gel, but he saw the band with silver. So he repeated that and cut off several (7-8) silver stained bands and the protein was identified.
Perhaps people from the mass spec service may help you in some way.
Good luck.
Do you mean he cut the same band but from several different gels? then it worked?
The mass spec people told me that they probabaly could not mass spec proteins with silver stain level, so i need more and more proteins.
I undestand you,
I had a similar problem some months ago (I wanted to identify an inmunoprecipitated protein) and still no solution due to the high amount of IgGs. It seems impossible. One guy from other lab told me that he sent to the mass spec service several bands, from different gels, of his silver stained protein. He was not able to concentrate enough to see it in one coomassie stained gel, but he saw the band with silver. So he repeated that and cut off several (7-8) silver stained bands and the protein was identified.
Perhaps people from the mass spec service may help you in some way.
Good luck.
Do you mean he cut the same band but from several different gels? then it worked?
The mass spec people told me that they probabaly could not mass spec proteins with silver stain level, so i need more and more proteins.
I have AB for my protein. the problem is i can not get enought proteins after purification to mass spec.
I undestand you,
I had a similar problem some months ago (I wanted to identify an inmunoprecipitated protein) and still no solution due to the high amount of IgGs. It seems impossible. One guy from other lab told me that he sent to the mass spec service several bands, from different gels, of his silver stained protein. He was not able to concentrate enough to see it in one coomassie stained gel, but he saw the band with silver. So he repeated that and cut off several (7-8) silver stained bands and the protein was identified.
Perhaps people from the mass spec service may help you in some way.
Good luck.
Do you mean he cut the same band but from several different gels? then it worked?
The mass spec people told me that they probabaly could not mass spec proteins with silver stain level, so i need more and more proteins.
Yes, the same band from different gels so he increased the amount of the protein he sent to mass spec. He was lucky, I tried and it didn´t work for me.
You may contact other MS service and ask them, they always ask for coomassie bands, easier for them to identify, but some people are able to identify silver bands.
Do you mean he cut the same band but from several different gels? then it worked?
The mass spec people told me that they probabaly could not mass spec proteins with silver stain level, so i need more and more proteins.
[/quote]
Yes, the same band from different gels so he increased the amount of the protein he sent to mass spec. He was lucky, I tried and it didn´t work for me.
You may contact other MS service and ask them, they always ask for coomassie bands, easier for them to identify, but some people are able to identify silver bands.
[/quote]
Do you mean he combined several same band from different gels, right? Does that make difference? I thought you bring more silver ions with more bands that is really what interfered with Mass spec?
Which country are you in I am in UK. I did contact several facility, one of them said they could have a go, then I send a band to them, nothing appeared. so I am kind of stuck. I wondered that should I contact more people?