BCA reagent composition?

Dr Engelbert Buxbaum engelbert_buxbaum at hotmail.com
Wed Dec 8 08:14:24 EST 2004


Wolfgang Schechinger wrote:

> 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), 

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

>I meanwhile have come across a website
> that tells you how to mix your own BCA protein reagent.
 
> Before I start testing the system by myself, I would like to know if anyone
> already has done so. 

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 ľl.

> BCA is the most expensive compound (even that Fluka -SAF-
> sells it at 92 Euro / 5gm).

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.


> 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?

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.



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