Too many problems in 2D - estimation? staining? running? (Nov/23/2007 )
Hi,
In my lab we've just started proteomics, so nothing much i know about this. First problem is estimation of proteins? I mean after precipitation we resuspend the protein in rehydration buffer which has urea, thiourea and chaps. Now how to estimate the protein? 
since these reagents interfere with lowry, bradford and BCA?? how do you guys do this?
Now second problem is I always get spots in lines? I mean when I see the gels in papers... the spots are quite random but I get it in linear fashion , why so? 
and I always get one vertical streak on the corner? don't know why 
my next problem is staining?this I've asked before also in this forum and got satisfactory answers but still I want to ask , Is there anybody who uses the silver stain, and cut the spots to detect on MALDI? how do you do that? and how much coverage you usually get?
hi
You can try doing silverstaining by a method described by Blum etal...It is MALDI compatible...we get good hits and we could identify proteins when using this staining... it is as good as colloidal coomassie
In my lab we've just started proteomics, so nothing much i know about this. First problem is estimation of proteins? I mean after precipitation we resuspend the protein in rehydration buffer which has urea, thiourea and chaps. Now how to estimate the protein?
since these reagents interfere with lowry, bradford and BCA?? how do you guys do this?Now second problem is I always get spots in lines? I mean when I see the gels in papers... the spots are quite random but I get it in linear fashion , why so?
and I always get one vertical streak on the corner? don't know why my next problem is staining?this I've asked before also in this forum and got satisfactory answers but still I want to ask , Is there anybody who uses the silver stain, and cut the spots to detect on MALDI? how do you do that? and how much coverage you usually get?
q1: you may run 2 precipitations: 1 for protein determination, the 2nd for 2D; however, as nearly all proteins should be precipitated, you may take the conc of your starting solution (lysate?)
q2: streaks: some proteins which are multiple posttranslational modified run in streaks; in your case you may think of overloading the IEF strip, or of incomplete separation
q3: silver stain should not interfere; we have done it with CBB or silver stained gel pieces or western blot (but of course not with bound antibody!)
q4: coverage: dependes especially on purity, alkylation, and amount
...how to estimate the protein?
For protein concetration determination, you can try to use this kit from Amersham Biosciences (GE Healthcare):
2-D Quant Kit
Accurately determine protein concentration in the presence of 2% SDS, 1% DTT, 8 M urea, 2 M thiourea, 4% CHAPS, 2% Pharmalyte™, and 2% IPG Buffer.
Quantitatively precipitates proteins while leaving interfering substances behind.
Linear response in the range of 0-50 µg protein, with recommended sample volumes of 1-50 µl.
hey guys thanks a lot for giving such nice suggestions 
. This was really helpful. We use vorum silver staining in the lab and that is also MS compatible, but this time I'll try to use blum silver staining. and this 2D quant is really good. Since I was searching for this kit on amersham's website , I came across with one good forum on proteomics, may be this wil help to all of you also. here is the link-
http://amersham.zeroforum.com