calculate the concentration First, prepare your working BCA reagent by mixing 10

Question

calculate the concentration

First, prepare your working BCA reagent by mixing 10 mL of Reagent A with 200 mu L Reagent B in a 15 mL conical tube (the resulting mixture can only be used for one day). If you require more reagent, prepare the amount using the same ratio of 50:1 A to B. Set up a series of wells in a 96-well plate (in quadruplicate) containing 0 (for the "blank"), 0.5, 1, 2, 3, 4 pg of BSA protein standard in a total volume of 40 mu L (no more than 40 mu L). Also prepare 4 wells containing appropriate amounts of your BSA sample of unknown concentration in a total volume of 40 mu L. Again, arrangement of your quadruplicate wells in a vertical column facilitates data movement in Excel. Add 200 mu L of working BCA reagent to each well with the multi-channel Pipetman, and incubate at 37 degree C for 30 min - place 96 well plate in the 37 degree C incubator, covered with Saran Wrap to prevent evaporation. The assay mix will turn pink with protein. Cool to room temperature and measure the absorbance at wavelength of 562 nm using the TECAN plate-reader and the "BCA Assay" option.

Explanation / Answer

Concentration of BSA will be :

1.0....blank

2.0.5 ug in 40 ul

=0.5/40 = 0.0125 ug/ul

3.1ug in 40 ul

= 1/40 = 0.025 ug/ul

4.2ug in 40 ul

=2/40 = 0.05 ug/ul

5.3ug in 40 ul

=3/40 = 0.075 ug/ul

6.4ug in 40 ul

= 4/40 = 0.1 ug/ul

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