What I do is load about 20 ug of protein on the gel, and next
to it I load 1/10 and 1/100 as much (serial dilutions). That way, I
can compare the combined intensities of any impurities in the 20 ug
sample to the intensities of the 1/10 and 1/100 "standards."You can
also load a 1/20 dilution, or whatever you want.
Many people simply run a sample on PAGE and then claim that
it's "pure" if they don't see any other bands; sometimes they even
claim ">99% purity" or the like. The problem I have with this is that,
especially with Coomassie staining, it is possible to destain the gel
long enough so that all of the faint impurity bands disappear. You can
make a sample that is, say, 75% pure appear to be "pure" if you
destain it long enough. In my view, you need to have some sort of
standards, such as the "10% impurities" and "1% impurities" standards
I described above, in order to make any quantitative claims at all
about purity. If you destain the gel for too long and you can't see
the 1% "standard" any more, then you can't claim 99% purity because
you can't detect an impurity level of 1%.
This is almost never done, however, mainly because, as one of
my former coworkers once told me, "nobody else does it that way."
Well, you wouldn't run a gel without molecular weight markers and then
claim a band is a certain molecular weight, would you?
Another criticism, and a valid one, is that the intensity of
Coomassie staining depends somewhat on the protein's amino acid
composition, so you can't be 100% sure that any impurities will stain
at the same intensity as your protein of interest. That's true, but
people often make claims about purity from Coomassie-stained gels
anyway, without running any purity standards. An imperfect technique
is better than a technique that has no calibration. If you want a
highly accurate technique, I would suggest using capillary
electrophoresis with a polyacrylamide matrix, or size-exclusion
chromatography. If you use the peptide bond absorbance at around 215
nm to detect, this should give accurate quantitation.
Note that silver-staining is not quantitative and should not
be used for estimating purity.
I hope this helps.
Matt
drabena at hotmail.com wrote:
>I am currently running SDS-PAGE's to calculate the "percent area" of a single protein. Can anyone tell me how to run this assay to determine the percent purity?
>
>http://www.biowww.net/index.php/forum/forumlist/1/
