SCOTT D. (gends at leeds.ac.uk) wrote:
: Hia,
: I need an accurate method of measuring protein concentration.
(stuff deleted)
I use Amido Black to measure protein concentration. This method is
more labour intensive then Coomassie but much more accurate.
It is described in Popov et al. Acta Biol Med Ger 34 p1441 1975.
Maybe you want to try it.
Solutions needed:
- Wash solution: 10% Acetic Acid, 90% Methanol
- Staining solution: 26mg Amido Black per 100ml Wash solution
- 0,1N NaOH
50ul protein extract
+300ul Staining solution
vortex
5min RT
centrifuge 4min at top speed
discard supernatant (be careful to not disturb the pellet)
Pellet + >=500ul Wash solution
vortex
centrifuge 4min at top speed
discard supernatant (carefully)
repeat washing steps until the supernatant becomes colorless
dissolve pellet in 350ul 0,1N NaOH (or 500ul)
measure at 615nm (I do it in 96-well plates in an ELISA Reader)
Care has to be taken not to discard a part of the pellet - especially
after the washing steps!
Standard deviations of 1% between doubles are possible.
Hope this helps
Klaus
Klaus Salger
Zoologisches Institut
AG MacWilliams
Luisenstr. 14
80333 Muenchen
Germany
Tel: ++49 89 5902-502
FAX: -450
e-mail: salger at zi.biologie.uni-muenchen.de
