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

2. 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

3. 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

4. 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

5. 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

6. Student Workstation Materials

7. Determining Protein Concentration
Play video: Bradford Assay

8. 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

9. 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

10. 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