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,
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?