how can I get enough proteins for Mass spec? drive me mad! - (Aug/06/2007 )
QUOTE (cathy @ Aug 6 2007, 04:50 PM)
QUOTE (K.B. @ Aug 6 2007, 06:10 AM)
You don't have antibodies for your target proteins, do you?
I have AB for my protein. the problem is i can not get enought proteins after purification to mass spec.
If you have some antibodies you can "spare" (or "waste"
) you can try immunoaffinity chromatography purification of your protein directly from cell culture medium or homogenate. It can work as single purification step or for concentrating your protein before actual purification. As good as I know from my labmate (guy with >30 years of lab experience) it works beautifully for low abundance proteins. -K.B.-
QUOTE (cathy @ Aug 6 2007, 05:17 PM)
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?
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?
If you put several silver stained bands together you are increasing the total amount of protein, that´s all. It doesn´t reach the coomassie level protein, but it worked for him.
I´m in Spain, check your PM please.
-Pumuki-
QUOTE (Pumuki @ Aug 6 2007, 08:25 AM)
QUOTE (cathy @ Aug 6 2007, 05:17 PM)
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?
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?
If you put several silver stained bands together you are increasing the total amount of protein, that´s all. It doesn´t reach the coomassie level protein, but it worked for him.
I´m in Spain, check your PM please.
I have done something like this. run several lanes of same sample on the same gel, then cut the whole several lanes of same band, send to ms. not working.
God help me
-cathy-