Question

Both Biuret and Bradford methods was used to determine unknown concentrations of both BSA and BIg.

The methods followed is described in detail in the Background section.

The following results was obtained:

Table 1: Absorbance at 595nm obtained for different concentrations of protein standards using the Bradford method

[Protein] (mg/ml)	0.00	0.125	0.25	0.50	0.75	1.00	1.50	2.00	Unknown
Abs. BSA	0	0.130	0.350	0.620	0.870	1.120	1.400	1.60	1.050
Abs. BIg	0	0.080	0.130	0.360	0.520	0.650	0.850	1.080

Table 2: Absorbance at 540nm obtained for different concentrations of protein standards using the Biuret method

[Protein] (mg/ml)	0.00	0.125	0.25	0.50	0.75	1.00	1.50	2.00	Unknown
Abs. BSA	0	0.026	0.045	0.075	0.111	0.128	0.172	0.218	0.26
Abs. BIg	0	0.024	0.052	0.092	0.136	0.162	0.223	0.264

Which standard curve would you use to determine the concentration of the Unknown protein sample?

		BIg standard curve using the Bradford method
		BIg standard curve using the Biuret method
		BSA standard curve using the Biuret method
		BSA standard curve using the Bradford method

Answer 1

1) Blg standard curve via biuret method

BSA Standard Curve using bradford method

2) 0.625 mg/ml protein is present in unknown sample

3) Average molecular wt of protein sample- 5782 g/mole or 5782 Dalton or 5.782 kDa

1 mole= 5782 gm

1 milimol = 5.78 gm or 5780 mg

therefore,

5780 mg= 1mM

4.4 mg = (1*4.4)/5780 = 0.000760 mM

or 7.6*10-4 mmol