"James Wee" <jywee at charter.net> wrote in message
news:vfn36p60ndkc8f at corp.supernews.com...
> 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 µg 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
>>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
