Team:Freiburg/Protocols/AmidoBlack
Amido black assay
Protein concentration Amido black assay
material: Amido black solution: 10% (v/v)Acectic acid, 90% (v/v)MeOH, 0.05% (v/v) Amido black; Destain: 90% (v/v)EtoH; 10% (v/v) Acetic acid …
duration: 1h
- transfer 20ul of protein extract into new Eppendorf tubes; pipet samples for the calibration curve (see table 1)
- add 1 ml amido black solution; vortex
- centrifuge for 15 min at max. speed, RT
- discard supernatant
- add 1 ml destain solution
- mix by inversion
- centrifuge for 15 min at max. speed, RT
- discard supernatant
- air-dry the pellet (takes about 15 min)
- add 1ml 0.2 M NaOH; vortex
- transfer the samplkes to disposable cuvettes
- measure the OD620
- calculate the concentration of your protein samples
| 1 µg/µl BSA | µg BSA |
|---|---|
| 5µl | 5µg |
| 10µl | 10µg |
| 15µl | 15µg |
| 20µl | 20µg |
| 30µl | 30µg |
