Cysteine / Cystine estimation in BSA (long(winded))

Josh jj_barnett at froggy.com.au
Sun Oct 21 18:54:01 EST 2001


"Artem Evdokimov" <AEVDOKIMOZ at cinci.rr.com> wrote in message
news:sWBA7.122191$6q.15854482 at typhoon.neo.rr.com...
> Dear Josh,
>
> It seems that you are confused. Let's see if I can make sense of what you
> wrote...

> Alternatively, if you did not load DTNB on the column, but rather tested
all
> the fractions that came out, then the higher activity in later fractions
is
> due to the fact that your beta-ME came out in the small molecule fraction
> too, so DTNB found lots of free SH to react with. Does that help ?

Yes, ran the column, collected the fractions (12), then tested these for A
412 with DTNB. The first few fractions had an absorbance close to zero,
while the rest had absorbances of around 1.6-1.8. This was the confusing
aspect in contrast to the Bradford samples where there was a definite "peak"
absorbance when the protein eluted.

> Read http://www.protocol-online.net/molbio/Protein/sh_quantitation.htm
>

> 1) you take your protein and reduce it with b-ME.
> 2) you run it down a sizing (in this case, essentially, desalting) column,
> in order to get rid of excess b-ME
> 3) use Bradford's test to determine which fractions contain protein, and
how
> much protein is there
> 40 use DTNB to determine the concentration of reducing groups, and then
> relate that to protein conc. thus obtaining the number of SH- groups per
> protein molecule.
>
> Hopefully, it helps. Read the original paper, mentioned in the URL above.

The bit I didn't get was to take an avergage absorbance!!!

Thank you very much indeed!!!!!!!!!!





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