Results of Micro-Bradford Preruns
by Eric, 3/29-30/99

I weighed 58.3 mg of BSA (Sigma A-7030, nominal 98-99% albumin,
purchased in 1986 and stored at 4 degrees since then) and made
a stock at 9.8 mg/ml in PBS + azide.

When diluted to nominal 0.42 mg/ml, the above stock had A280 of
0.248 (checked on 2 specs), which at 0.67/(mg/ml) gives 0.37
mg/ml. This is 88% of nominal, which is reasonable considering
that no attempt was made to dehydrate the powdered protein before
weighing it.

Standard curves (done several times) were reasonably
reproducible, showing that the protein concentration detectable
at the minimum reliable absorbance of 0.1 is between 2 and 5
micrograms/ml, the less sensitive value being for wells which
have sat for 30-60 min after mixing, or old batches of Bradford
reagent (I tested one from 1992 which gave slightly lower
absorbances than a newer batch, BioRad lot 58731A).

My standard curves were fairly linear to corrected absorbance
0.4, curving down a bit after that.   Corrected absorbance 0.3
occurred at about 20 micrograms/ml.

The blank (160 microliters of water + 40 microliters of reagent)
had A620 of 0.4.  Thus, if a total absorbance of 1.0 is the
maximum considered reliable, the maximum corrected absorbance
allowed should be about 0.6, which was achieved with about 50
micrograms/ml BSA.

The 96-well plate readers give readings close to those obtained
with a 1 cm path in a spectrophotometer.  Since the path length
for 200 microliters in a well is about 0.7 cm, the results are
probably 'corrected' with a factor of about 1.4.  The older
reader has a correction factor dial which was found (and used)
set at 1.1.

After mixing, the absorbance gradually declines (contrary to what
the manufacturer states) to about 65% of its initial value within
an hour.  After sitting overnight (not recommended, just
curious), the wells contain blue precipitate.

If the absorbance with the new reader (nominal 620 nm) is taken
as 100%, the old reader gives 107% at nominal 570 nm, and 78%
at nominal 630 nm.

To my surprise, a solution of 62 micrograms BSA/ml in a 12 x 75
mm polystyrene test tube, stored overnight at 4 degrees, did not
decline in concentration significantly. Thus a 1 mg/ml stock
should be stable for a week or so (azide present, at 4 degrees).
I would suggest giving the students 1 mg/ml stock, and letting
them dilute it 20-fold to the desired 50 micrograms/ml for doing
the standard curve.

Mixing by 8 in and out's was confirmed to be adequate under the
conditions recommended by watching mixing of trypan blue into