densitometric analysis for silver stained/coomassie gels? - is it done? staining efficiency same for different proteins? (Oct/20/2009 )

hi all..
i m doing minor impurity analysis for my purified protein preparation by sds-page or ief gels. i use either silver or coomassie staining for detection. i may have related impurities like the truncated forms or aggregates or charge variants of my protein in near 0.5 or 1% amounts. i run a 0.5% and 1% solution of my purified protein where i see a main band, for comparison of impurity band intensity.
i wish to know if i can do densitometric analysis for such a purpose.

is this applicable to silver stained gels and or coomassie gels?
the question also is that are the staining efficiencies for any of these stains considered to be different for different proteins? if yes, why so? can such an analysis be done then?

thanks.

you can use densitometry for coomassie stained gels and will be able to determine approximate relative concentrations for all proteins in the gel.

with silver stain you can only determine relative concentration for the same protein (or fragments) with any degree of confidence.

mdfenko on Oct 20 2009, 09:43 AM said:

can u pls explain on why u say so. thanks.

silver stain is not quantitative across different proteins. it can be quantitative for the same protein up to a point that you will need to determine by running a standard curve.

is this what you need?

mdfenko on Oct 21 2009, 08:18 AM said:

thanks! but i need to understand why u say so.
as i understand coomassie staining depends on the basic residue (lysine, arginine) content of a particular protein. hence, this staining efficiency should be expected to vary across different proteins. but from what i read, coomassie is not expected to differ much. while silver stain is. i need to understand principally why this is so. thank you..

if i understand right... there can be more variation even for the same protein in silver stained gels from gel to gel as it involes lot of reagents and the preparation of the reagents each day will vary leading to the change in intensities. Ya for the same gel it might be fine by using some kind of standard concentration which can be the case with comasssie too.. but coomasie wont give such a variation with the same protein atleast over different gels!!!
(I am not sonsidering the gel preparation and runnign errors!!!)

coomassie g-250 is more specific than coomassie r-250. with r-250 you will get a more uniform stain and will be reasonably quantitative across a range of proteins.