Triton X-100, TCA, Protein determination

Anne Spang spang at vms.biochem.mpg.de
Tue Jul 11 14:32:48 EST 1995


Hi!
Since I received quite a number of e-mails, here the protocol of the Amido
black stain of proteins

- make the samples up to 0.25 ml containing 1-10 ug of protein.

- add 30 ul 1 M Tris-Cl pH 7.5, 1% SDS

- add 70 ul 50% TCA

- incubate 2 min RT

- assemble Schleicher and Schuell dot blot apparatus (BioRad filter
apparatus is doing the same job): 1 Filterpaper soaked with 5% TCA,
nitrocellulose also soaked with 5% TCA

- rinse each well with 400 ul 5% TCA, apply vacuum

- spot samples, apply vacuum

- rinse each well with 400 ul 5% TCA, apply vacuum

- disassemble dot blot apparatus, stain the nitrocellulose for 2 min in
0.1% w/v Amidoblack (MeOH:HAc:H2O = 45:10:45)

- rinse in destilled water

- destain 3x1 min in MeOH:HAc:H2O = 270:6:24

- rinse in destilled water, dry the NC on Whatman filter

- cut out spots, place in glass tubes, add 200 ul elution buffer (25 mM
NaOH, 50uM EDTA, 50% EtOH)

- incubate 20 min at RT

- read at 630 nm (for ELISA: spot the 200 ul in a microtiter plate)


   Hope this helps!!
      Anne



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