BETA ACTIN?????

Ralf Breuker Ralf.Breuker at ruhr-uni-bochum.de
Wed Oct 21 01:35:03 EST 1998


Dear Dave,

Dave O'Neill schrieb in Nachricht <36267150.3A4EDDCD at julian.uwo.ca>...
>HI,
>Due to technical problems (received in sample buffer)  I was unable to


That is no technical problem. Of course, Bradford doesn't work with sample
buffer, but try another protocol.

We work with amidoblack. Try the follwoing protocol:



Solutions:
Dye solution:
240,0 mg  Amidoblack 45,0 ml  MetOH  45,0 ml  H2O  10,0 ml  CH3COOH
Destaining:
40% Methanol, 10% Acetic Acid in bidest.
Elution:
80,0 ml  CH3COOH  10,0 ml  HCOOH 10,0 ml  H2O  1,0 g  TCA

Pipet 10 microliters of the diluted proteins (even in SDS sample buffer) on
a sheet of cellulose acetate. Dry extensively, stain for ten minutes in Dye
solution, destain until background has vanished. Change destaining solution
several times. Dry again extensively. Cut protein dots out and resolve them
completely in 3 ml of elution solution. (Is a bit tricky, I confess).
Measure extinction in a Photometer at 630 nm.

The extinction values are proportional in a range between 0,1 and 3 mg/ml
Protein. You need of course a standard curve for protien determination.
Fortunately, results are very reproducable, so you don't need to make the
standard curve more than once.

Bibliography

1. Schaffner, W. & Weissmann, C. Anal. Biochem. 56, 502-514 (1973).


Good luck

Ralf








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