- add to 10 ul sample 1 ml
alkaline copper reagent and vortex
- incubate 1 h at rt
- add 50 ul folin-ciocalteu
phenol reagent
- incubate 45 min at rt
- measure absorbance at
750 nm
- make BSA standard curve:
0 to 100 ug per 10 ul PBS
- fit standard curve with
second order polynomal
- OD660/5 ~ mg/ml protein
in E. coli culture
- e280 (BSA) =0.667 ml/mg/cm
alkaline copper reagent: 100 parts A and 1 part B
A:
2% Na2CO3
1% SDS
0.4% NaOH
0.16% sodium tartrate
B:
4% CuSO4.5H2O
- spot 5 ul protein sample
on nitrocellulose (NC)
- air-dry for 5 min
- immerse in stain solution
for 3 min
- wash 2x 3 min in water
- wash 2x 3 min in wash
solution
- wash 5 min with water
- air-dry 5 min
- elute stain with 1 ml
elution solution (while shaking the sample)
- measure absorption at
630 nm of the eluant
stain = 0.1 % amidoblack,
45% methanol, 10% acetic acid
wash = 90% methanol, 2%
acetic acid
elution = 50% ethanol, 50%
50 mM NaOH/0.1 mM EDTA
- use BSA solutions (0 to
5 mg/ml) for calibration
- this protocol also works
for proteins in SDS-PAGE buffer