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
