BCA reagent composition?

Hi folks,

I would like to homemake BCA reagent and adapt the assay to microplates.
Although everybody just buys the Pierce stuff and honestly cites Smith et
al (1985, not available online), I meanwhile have come across a website
that tells you how to mix your own BCA protein reagent.

They use 10gm of BCA per liter in a
pH 11.25 buffer containing
20g/l sodium carbonate
1.6g/l sodium tartrate
9.5g/l sodium bicarbonate
adjust pH with NaOH

Solution B is 4% m/v CuSO4 (pentahydrate) in water.

BCA soln and soln B are mixed in a 50:1 ratio (v/v), added to an equal vol
of test solution, heated to 60 degC for 30 min and then read at 562nm.

This means, I'll have 100mg BCA per ml while my protein solution I need to
measure is 10 to 100 *micro*gram per ml.

Before I start testing the system by myself, I would like to know if anyone
already has done so. I'd like to find out if 1 gm BCA per liter is
sufficient as BCA is the most expensive compound (even that Fluka -SAF-
sells it at 92 Euro / 5gm).

Thanks for your input and best regards,

Wolfgang

PS - what is tartrate actually for? i remember it from fehling's solution -
is it involved in the reduction of Cu(II) to Cu(I)? Or does it simply form
another nice but colourless complex with CuII to mask it?
---

Wolfgang Schechinger wrote:


But surely your library will have a subscrition to Anal. Biochem.?



The assay works very well in microplates, incubation can take place in a
37 degree incubator or water bath. Just scale down all volumes to a
total of 200 µl.


Make sure that your reagent is pure enough, you need either BCA
specified for protein determination (we always got it from Pierce) or
you have to recrystallise, the recipe is in the Smith paper, IIRC.
However, given the inevitable losses during recrystallisation and the
working time involved, buying proper reagent quality is probably cheaper
over all.



Yes, the Cu(II) in the reagent is reduced to Cu(I) in the biuret
reaction with the peptide bond, forming a bluish-purple complex with the
protein. Tartrate keeps the Cu(II) in solution, under the alkaline
conditions it would otherwise precipitate as Cu(OH)2 or CuCO3.

The detection limit of the biuret reaction is low, so Smith et al
suggested to detect the resulting Cu(I) with BCA, improving the
sensitivity of the assay by 3-4 orders of magnitude. Note that the
well-known Lowry et al. procedure works along the same line: Cu(I)
reduces the phosphomolybdic/phosphotungstic acid in the Folin-reagent,
forming a deep blue complex.