Removal of protein from glycogen extractant?

Ngo Thi Thuy Huong nt_thuyhuong at yahoo.com
Fri Mar 17 03:20:05 EST 2006


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...?

Thanks,

Twince

> 
> 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
> protein/glycogen
> 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
> http://www.bio.net/biomail/listinfo/methods
> 




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