thank you so much for that information. Regarding stoichiometry and quantification, we were hoping that band intensities of the protein of interest using western techniques, and subsequent densitometric quantification of the bands of interest would help us to quantify the protein levels in each extract. But western techniques for transfer of such small proteins proved to be inconsistent across the blots (the protein binding was not always uniform across the blot, and thereby producing bands which were not always related to the protein quantity), which I learned later was an inherent problem in all western blots. This caused a significant amount of error which masked the protein quantity variations among the extracts. By doing a large number of standardization experiement, I was able to reduce this variations in western transfer to the least possible extent.. but still not good enough to detect small variations in protein content.
I am a novice in the field of S35 labelling, though I am not short of experts in that field around me. Could you give me a gist of how you do an invivo labelling of the protein (by the way, the source of the protein are human cells which we culture in our lab), so that only the protein of interest is tagged. Any websites or papers regarding this would also be very useful.
thank you so much for your interest in my problem
You will not be able to label only the protein of interest in vivo. If your
protein of interest is somehow "inducible" with a treatment, then it should be
possible to selectively allow more label to be in corporated into it during the
treatment interval. As I said earlier, if you are able to identify the
polypeptide in situ (gel) through staining, then you will be able to show the
variation in the intensities of the bands across the autoradiogram.
The labeling in vivo is not difficult at all and it is done by many in the
field of gene expression. The label is usually [35S]-Met and is added to the
the growth medium when desired. You can also produce clean labeling by chasing
the label off with cold Met for a specific time after you withdraw the
induction condition from the cells. For animal cells, the uptake is much
faster and works a lot better than what I was used to (Plants and fungi). There
are a lot of papers out there that you you can refer to for a complete
protocol. Check out the Meth Enzymol also. While it is/was a very widely used
method, it does need sandardization and adaptation for the investigator's
particular system. Since it will be a direct measure from the gel itself, it
will obviously be better than having to quantitate from another matrix (viz.
western blot). For quantitation through this, you will still need to devise a
direct stochiometric method through relationship with a known protein
(inducible and/or housekeeping) and I still can't quite figure out how. I
doubt that an absolute quantitation of any protein without purification is
Let me know if I missed anything.
Quoting "Jayakumar, R" <[Only registered users see links. ]>:
Hiranya S. Roychowdhury, Ph.D.
Coll. Asst. Professor,
Dept. of Chemistry & Biochemistry
Rm# 336, Chemistry Bldg.; MSC 3MLS
New Mexico State University
Las Cruces, NM 88003
Metabolic labelling affects all proteins made by your cells during thee
labelling periode, there is no way to limit inclusion of label to
specific proteins (unless you use /in vitro/ translation and have only
one mRNA). But if you have an antibody against your protein, you could
use immunoprecipitation for a one-step purification. The immunocomplex
can be fished out of the sample with Pansorbin (Calbiochem, a
preparation of dead Protein A producing bacteria. They have a booklet on
experimental technique). If you optimise the conditions carefully, you
can quantitate your protein by counting the precipitate.
The techniqque of metabolic lebelling is simple, the growth medium is
exchanged against one that doesn't contain Cys and Met (this is
commercially available). Instead, you add a mixture of 35S labelled cys
and met ("Translabel"). The first time you do this in a small batch,
taking aliquots every 15 min or so to check when the highest level of
your labelled protein is produced. Once that has been established,
change the labelling medium against one with plenty of cold cys and met
and incubate for a chase periode of 15-30 min, to make sure that all
labelled protein has come of the ribosomes. Then homogenise and purify.