Question

Experiment 8: Bradford assay and UV-method for determination of protein concentrations Materials - An ice bucket with ice, your purified sample, three visible plastic cuvettes for Bradford assay, one UV plastic cuvette for UV-method, a piece of parafilm, a Vis-spec, a Lab Quest, Bradford reagent, white tape (share), a sharpie. Bradford assay (reference 21) 1. Add 990 uk Bradford assay reagent to a plastic cuvette 2. Blank at 595 nm 3. Add 10 ml of purified protein to the plastic cuvette containing Bradford reagent 4. Use parafilm to mix contents in cuvette 5. Allow color to develop for 5 minutes (no longer than 10 min) 6. Measure absorbance at 595 nm three times. 7. Calculate the concentration of your sample based on calibration curve with BSA as a standard (Figure 1). Record. y 0.96 0.0086 Figure 1. Bradford standard.curve the standard surve was made recently Reference 1. Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal Biochem 72,248- 254.

I forgot to put down that the observance (=OD) at 280nm is 0.679A

Answer 1

A280 = 0.679

ε280 = unknown as the standard curve is at 595nm

c = 1.702 μg/μL [Since, there is 1.702μg of BSA (Protein) in 1μL of the solution]

l = 1cm

Hence the only unknown entity is  ε280,

ε280 = A280/(Cl) = 0.679/1.702 = 0.3989

Note: As far as my knowledge goes, the standard curve should pass through the origin as the absorbance should be zero when the concentration is zero, which is the entire point of using the blank. Additionally, the R value should be closer to 1, even though 0.987 is in no way bad, I have gotten better results while conducting experiments in the laboratory myself so you might want to redo the standard curve for the experiment to be truly accurate.