Leslie-
A good way to get % purity is to load a gel with a series of amounts, so
that you're in the linear range for each of the species of interest. Plot
the area vs amount loaded for each species of interest in its linear range,
and calculate the slope. Use these slopes (instead of individual peak
areas) to calculate purities or ratios of species.
Vernon A. Shoup
Regeneron Pharmaceuticals
Rensselaer, NY 12033
(518)488-6012
vernon.shoup@regpha.com
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Date: 1/13/98 7:41 PM
From: Leslie J Novick
I am trying to quantitate impurities in "pure" protein by SDS PAGE Commassie.
I am using pharmacia scanning software (ImageMaster) with gels scanned using
LabScan in Transmittance mode. The 'pure' proteins are glycosylated (thus not
sharp bands), the gels are gradient and the impurities are <5%.
Issues I am addressing include:
1) Scanning parameters (300 vs 600 dpi)
2) Lane drawing: Should the lane encompass the whole protein, or would a thin
lane through the middle work better?
3) Coomassie staining: a) Do glycosylated proteins abs. coomassie differently
than non-glycosylated proteins? b) Why do I find that lower molecular weight
protein standards absorb coomassie MORE than higher molecular weight proteins?
c) If a gel is well destained, do I need to consider the acrylamide gradient?
4) Linear range: to get the contaminants within their linear range I must
overload the protein. By doing this, the calculated % purity is wrong.
Whatever method I use would be for an FDA submission. Does anyone have
any ideas or references about this?