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Lowry protein determination

Lowry protein determination - Protein Forum

Lowry protein determination - Protein Forum


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  #1  
Old 06-26-2003, 04:21 PM
James Wee
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Default Lowry protein determination




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  #2  
Old 06-27-2003, 03:29 AM
James Wee
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Default Lowry protein determination

I use OD 750 to read cellular protein concentration of a bacteria. I don't
use Coomassie reagent, instead, I use Folin reagent as the stain. I use 30,
60, 90 and 120 g of BSA to construct the standard curve. It doesn't sound
right to substract 0.248 from all sample readings. Some of the sample
readings will go below 0, though the curve goes through 0. Could u explain
more about over the linear range of detection?

Thanks,
James


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  #3  
Old 06-27-2003, 04:01 AM
EK
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Default Lowry protein determination


"James Wee" <[Only registered users see links. ]> wrote in message
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30,
You also should use 0 ug as one of the standard concentrations and subtract
that from your 30, 60, 90 and 120 ug ones. I would also add something like
0.5, 2, 5, 10 and 240, 500 ug just to see where are the boundaries of the
linear range in your particular setup.

Linear range of concentrations is where the dependence of optical density on
the protein concentration/content can be closely approximated by a linear
function. If you go over or below that range, the dependence does not have a
linear character anymore and thus cannot/should not be used for estimations.

What is the source of your Folin reagent and is you method based on a
standard Folin-Chiocalteau procedure (or Lowry method) or some modification?
Do you have a manufacturer's manual? Can you get it on the web? I am not
sure that 750 is the right OD for Lowry, 660 sounds closer to my heart.

Also, please be aware that some compounds interfere with detection by Lowry
method. These compounds, among well known ones, are (!) Tris, HEPES, and
other zwitterionic buffers, EDTA, some sulfur compounds (e.g., sulfides), so
that you need to think about removing those from your protein solution by
e.g. dialysis or precipitation with e.g. 7% trichloroacetic acid followed by
resuspension in 0.1N NaOH and neutralization (same procedure is recommended
for standard protein). The presence of interfering compounds would explain
why

As a general remark, I believe Lowry method is much more cumbersome and not
so sensitive as Bradford (Coomassie) that I would recommend you for its
relative low cost, speed and simplicity. To compare, Lowry method requires 2
reagents and about 50min total incubation time before reading, whereas
Bradford requires only about 5 min.

-Emir


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  #4  
Old 06-27-2003, 03:41 PM
Dr. Hiranya S. Roychowdhury
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Default Lowry protein determination

Coumassie G is the reagent for Bradford's assay, and Lowry's assay uses the3
Folin's. So, there is not much of a deal there. Your spec reading for the 0
BSA, no matter what it is, should be used as the baseline. In other words, that
IS THE starting point, the zero. The spectrophotometer has to be "Zero'd" with
the 0 BSA tube (which also means the "Water Blank"). I cannot fathom where the
confusion is here! The Lowry assay is prone to interference by a lot of
different things that may be there in your extraction buffer, so, it would be a
a great idea to use the extraction buffer instead of water for the blank
("Buffer Blank").
As someone else also suggested, you may be better of using the modified Lowry
assay called the BCA assay (sold by Pierce). You may also try Bradford's assay
which seems to be best suited for samples containing interfering salts.

Hiranya



Quoting James Wee <[Only registered users see links. ]>:



--
Hiranya S. Roychowdhury, Ph.D.
Coll. Asst. Professor,
Molecular Biology,
Dept. of Chemistry & Biochemistry
Rm# 336, Chemistry Bldg.; MSC 3MLS
New Mexico State University
Las Cruces, NM 88003
---
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  #5  
Old 06-27-2003, 03:46 PM
Dr. Hiranya S. Roychowdhury
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Default Lowry protein determination (follow up)

BTW, ar you sure Lowry's needs 750nm? I haven't used Lowry in a long time, but
if my memory serves me right, the absorbance is measured at around 600nm!


Quoting James Wee <[Only registered users see links. ]>:



--
Hiranya S. Roychowdhury, Ph.D.
Coll. Asst. Professor,
Molecular Biology,
Dept. of Chemistry & Biochemistry
Rm# 336, Chemistry Bldg.; MSC 3MLS
New Mexico State University
Las Cruces, NM 88003
---
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  #6  
Old 07-03-2003, 07:35 AM
James Wee
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Default Lowry protein determination (follow up)

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



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  #7  
Old 07-03-2003, 10:50 PM
EK
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Default Lowry protein determination (follow up)


"James Wee" <[Only registered users see links. ]> wrote in message
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NaOH,
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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  #8  
Old 07-04-2003, 08:06 PM
EK
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Default Lowry protein determination (follow up)

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.
[Only registered users see links. ]
-Emir

"James Wee" <[Only registered users see links. ]> wrote in message
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tightly
from
a
presence


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  #9  
Old 11-11-2008, 09:35 PM
Pipette Filler
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Default Re: Lowry protein determination

I have a problem about my viral antigen with PEG-6000. I have a semipurifyied viral antigen and I used PEG after that to determine protein concantration I used Lowry, but viral protein with Folin didnt emulsified and didnt give an absorbance value. I USED TES BUFFER TO prepare PEG.EDTA TRİS and NaCL to prapere TES BUFFER. I have 50 ml viral antigen purified with PEG with that TES BUFFER. I wonder if this buffer contain or PEG effected or interfereted Lowry. How can I measure this protein. Can I clean this chemicals from my viral antigen. Please help meeeeeee.
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  #10  
Old 11-11-2008, 09:50 PM
Pipette Filler
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Default Re: Lowry protein determination

I have a problem about my viral antigen with PEG-6000. I have a semipurifyied viral antigen and I used PEG after that to determine protein concantration I used Lowry, but viral protein with Folin didnt emulsified and didnt give an absorbance value. I USED TES BUFFER TO prepare PEG.EDTA TRİS and NaCL to prapere TES BUFFER. I have 50 ml viral antigen purified with PEG with that TES BUFFER. I wonder if this buffer contain or PEG effected or interfereted Lowry. How can I measure this protein. Can I clean this chemicals from my viral antigen. Please help meeeeeee.
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