"Kyle Legate" <legatek at sympatico.ca> wrote in message
news:apjKa.4884$OE2.529926 at news20.bellglobal.com...
> Charter News wrote:
> >
> > I have a problem to construct a standard curve by using Bovine Serum
> > Albumin. The standard curve is linear, but it intercepts y-axis at
> > 0.248 Abs (Beckman spectrometer). The sample values I want to compare
> > with the standard curve are lower than the y-intercept value, e.g.
> > 0.0987. Is that the problem with the BSA? Or there is something wrong
> > with my sample readings?
> >
> Do you mean that your blank with no protein gives a reading of 0.248? When
I
> make a standard curve I always blank the instrument with a sample
containing
> no protein (try to keep buffer components constant between your standadr
and
> your sample to eliminate the effect of these components on the colour
> development); this ensures that the standard curve goes through 0. In any
> case it doesn't sound like you have much protein in your sample. Perhaps
try
> a more sensitive technique (BCA assay microtitre plate method?)
>>0.248 sounds like a blank reading of OD595 for me. Subtract that value from
all samples if you did not do that. If you do that and get the intersection
at 0.248, try producing another standard curve with lower concentrations of
BSA in addition to ones you have used in your previous experiment. Se how
the curve goes. It might be that your are going over the linear range of
detection with concentrations you have used. Check with the provider of your
Coomassie reagent on the detection range (or read it the manual:-)). Also,
don't use 1x Coomassie mixture that is older that 2 weeks.
Emir
