Removal of protein from glycogen extractant?
zebedeeboy at hotmail.com
Fri Mar 17 20:06:29 EST 2006
The Phenol-sulfuric acid assay is a bit tempermental. To get it to work
properly you need to shoot the conc sulphuric acid directlty onto the
sample. I used to use polycarbonate LP4 tubes and you could tell when it had
worked properly because the tubes would melt.
"Ngo Thi Thuy Huong" <nt_thuyhuong at yahoo.com> wrote in message
news:mailman.201.1142612506.16885.methods at net.bio.net...
> Thank you very much Dr. Engelbert Buxbaum for your
> kind response. The üiüetting might contribute to the
> problem I met, I agree. But I think it is not the only
> source because I have been used to this kind of work
> and at this time I also do protein quantification and
> I do not have any problem with this method, the STDEV
> is very small, less than 2%.
> I used phenol-acid sulfuric acid method for glycogen
> determination. With this method, it did not mention
> the removal of protein from the samples. I worked with
> 7 standards and 4 replicates and I think it is
> statistically fine. But nevertherless, I sill had very
> high STDEV and I think it caused by the present of
> suspended protein in the solution and when pipetting I
> might get different protein amount into microwell.
> This resulted in different OD.
> Then I red a basic book about carbohydrate
> quantification and I found that protein might be
> contribute to the high STEDV and I tried to remove
> protein by the two methods which I mentioned in the
> first letter. The hot ethanol worked better than cold
> ethanol method. Briefly, I digested the sample with
> KOH 30% in hot water bath shaker (at 95°C). Then
> absolute ethanol was added (final conc of ethanol was
> about 70%) and put in hot water bath shaker (95°C for
> 15 min). Samples then vortex and centrifuge at 8000 xg
> for 15 min to discard the precipitates. The sample now
> are ready for glycogen assay with phenol-acid sulfuric
> acid method.
> I would be highly appreciated if you have any idea how
> I can improve the protein removal method, e.g.
> centrifuge force? ethanol conc? heating time...?
>> Are you sure it is not simply your pipetting? No
>> offence intended, but
>> it takes months of practice until one can reliably
>> pipet to a standard
>> deviation of 2% or less. And your r^2 can easily
>> happen if you only have
>> a few data points.
>> If you have always the same protein composition and
>> ratio, the presence of protein should not increase
>> standard deviation
>> (although it may affect the correctness of the
>> result), unless the assay
>> design is faulty (e.g. protein precipitates are not
>> removed before
>> photometric reading). Can you tell us which method
>> you are using?
>> Methods mailing list
>> Methods at net.bio.net
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