Lowry protein determination (follow up)

EK nobody at elnino.com
Fri Jul 4 15:06:15 EST 2003


As far as I remember the Lowry method, you do the following.
1) Mix NaOH, CuSO4+NaKTartrate+ and 2%Na2CO3 to make reagent 1.
2) Reagent 2 is Folin phenol reagent.
3) You add an aliqiote of protein in a fixed volume.
Your blank is where you add the same volume of water, or even better buffer
in which your proteins are dissolved INSTEAD of your protein solution. Then
you add reagent 1, mix and wait, add reagent 2, mix and wait again, read the
samples.

Do the search in Google(.com) for Lowry protein assay. Here is what I just
found for you this way.
http://bric.postech.ac.kr/protocols/general_methology/general_protocol_76.html
-Emir

"James Wee" <jywee at charter.net> wrote in message
news:vga2hhe62t3dc3 at corp.supernews.com...
> Thanks. I prepared my blank by adding NaOH first, adjust it to 1mL using
> distilled water. Secondly, I added CuSO4+NaKTartrate+ 2%Na2CO3, and Folin
> reagent the last. Before I took the reading of the samples, I blanked the
> Beckman spectrophotometer using the blank solution have I prepared. Thus,
> all the sample readings are adjusted. Could this be a problem?
>
> James
>
> "EK" <nobody at elnino.com> wrote in message
> news:3r2Na.52$Y4.12560 at news.uchicago.edu...
> >
> > "James Wee" <jywee at charter.net> wrote in message
> > news:vg7bqu5sp0r973 at corp.supernews.com...
> > > Thanks for your comment. I checked through all my reagents and found
> that
> > > there is something wrong with IN NaOH that I used. After using the new
> > NaOH,
> > > the readings are low now. It intercepts with y-axis at  0.04. I wonder
> how
> > > the NaOH solution (which contains more NaOH) makes the color darker.
> > >
> > > James
> > >
> > >
> > >
> > Make your NaOH at least once every couple of weeks and keep it in
tightly
> > closed bottle, or use a stopper with CaOH trap. NaOH will absorb CO2
from
> > air to form insoluble Na2CO3. Also, some microorganisms can live in
> > solutions of such a strength(plus the strenth will be reduced in time as
a
> > result of growth). Your high values are most probably due to the
presence
> of
> > growth of contaminating species that as you can guess are made of
> proteins,
> > too. By the way, I still don't understand how your blank would not be at
> > zero. Do you compare your values for blank with numbers someone gave you
> as
> > a guideline?
> > -Emir
> >
> >
> >
>
>





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