Protein Assay
Description
In this interactive lab, postgraduate students perform a complete Bradford protein assay from first principles to final result. After exploring why Coomassie Brilliant Blue's absorbance shifts from 465 nm to 595 nm upon binding protein, students calculate and prepare their own BSA dilution series using the C₁V₁ = C₂V₂ relationship, then pipette sample, water, and Bradford reagent into each tube themselves and watch the color develop in proportion to protein concentration. Students operate a virtual spectrophotometer end to end, selecting the wavelength, warming up the instrument, blanking it, and reading each standard and unknown sample, before building their own standard curve, fitting it to y = mx + b, and calculating unknown protein concentrations from their data. Additional unknown samples let students repeat the full technique independently, reinforcing the workflow a real quantitative protein assay demands.
Learning Outcomes
By the end of this lab, students will be able to explain the principle behind the Bradford assay and compare it with UV absorbance, Lowry, and BCA methods for protein quantification, and apply the C₁V₁ = C₂V₂ relationship to calculate and prepare a dilution series of protein standards. Students will also be able to correctly operate a spectrophotometer, including selecting the appropriate wavelength, blanking the instrument, and reading absorbance values, and construct a standard curve to derive a best-fit equation relating absorbance to protein concentration. Finally, students will be able to calculate unknown protein concentrations from measured absorbance values, recognize when a sample falls outside the assay's linear range, and identify common sources of error and interference in colorimetric protein assays.