Filter dye-binding method for protein determination
(KO/AG (?) handwritten)
Materials:
 Whatman No.1 filter paper
 0.25% CBB in 7% CH3COOH (filtered) or regular CBB solution
 MeOH
 7% CH3COOH (destain solution)
 66% MeOH + 1% ammonia (extraction solution)
 BSA standard
Method:
1. Draw grids on Whatman No.1 paper with pencil (1.5cm for 10µl sample, 1cm for
5µl sample)
2. Spot your sample and BSA control onto each square. Let it dry.
3. Drip in MeOH to fix protein and rinse off BPB for 10-20 sec.
4. (optional) Allow filter to dry.
5. Stain with CBB for 15 min.
6. Destain with 7% acetic acid. Change the solution frequently for faster destaining.
Do not leave overnight!
7. Dry the filter paper briefly (it does not have to be completely dry).
8. Cut out each square and put into test tube. Add MeOH/ammonia solution – use
2ml for 1.5cm square, 1ml for 1cm square.
9. Vortex and let sit 5min., vortexing occasionally for dense stain.
10. Put aliquot in cuvette, measure OD605 (595-610 range okay).
11. Draw standard with BSA and determine protein concentration.