Protein Structure and Analysis Activity 7.1: Protein Quantitation Using the Bradford Assay

Activity 7.1: Protein Quantitation Using the Bradford Assay Research question: How much protein is present in milk? Objectives: Perform a Bradford assay on two milk samples Perform a Bradford assay on a series of standards of known protein concentration Use a spectrophotometer to measure the absorbance of Bradford assay samples Generate a standard curve of protein concentration against the absorbance at 595 nm Determine the protein concentration in the milk samples

Safety Reminders Follow all laboratory safety procedures Read and become familiar with MSDS for the Bradford reagent Wear appropriate PPE Bradford reagent can stain the skin and clothes

Skills to Master Perform a Bradford assay Use a spectrophotometer (Activity 5.4) Generate a standard curve using absorbance values Determine experimental values using a standard curve

Tips Mix samples thoroughly with the Bradford reagent Pipet up and down or cover with Parafilm and invert to mix Samples should be incubated at room temperature for at least 5 min after mixing with the Bradford reagent Orient cuvettes correctly in the spectrophotometer so that light passes through the sample Do not handle the cuvette where light passes since fingerprints can interfere with the reading If a spectrophotometer is not available, visually compare the samples to the protein standards

Student Workstation Materials

Determining Protein Concentration Play video: Bradford Assay

Protocol Highlights/Tips Make sure samples are mixed thoroughly Touch the cuvettes only at the top Make sure the cuvettes are oriented properly when placing them into the spectrophotometer Ensure that the samples and controls have incubated 5 minutes after mixing with the Bradford reagent before analysis Compare unknown samples to controls by eye before using the spectrophotometer

Gel Results Compare the samples by eye to the standards Spectrophotometers can be used to take absorbance readings at 595 nm Graph the controls and use to estimate unknown absorbance

Summary Make sure to: Record all steps in your notebook Be careful not to touch cuvettes where light passes through Mix samples thoroughly Compare visually the known and unknown samples that have been mixed with Bradford reagent