The Bradford Assay
Lecture 20
How do you figure out how much
protein you have in a sample?
What if you found a chemical that
changes color when you add it to a
protein solution?
Brilliant Blue G
Brilliant Blue G or Coomassie Blue
• Absorbs light at 465 nm
• When protein is present it sticks to the
protein and absorbs light at 595 nm
Bradford Assay
1. Make solutions of a known protein
(usually bovine serum albumin or BSA)
2. Mix a sample from each solution with the
Bradford reagent (Brilliant Blue G +
Phosphoric acid + Methanol)
3. Measure absorbance at 595 nm
4. Plot on a graph
5. Use graph to determine unknown protein
concentration based on absorbance.
Bradford Assay
1.2
1
Absorbance 595 nm
0.8
0.6
0.4
0.2
0
0 0.2 0.4 0.6 0.8 1 1.2
Protein Concentration (mg/mL)
Add Trendline and Calculate
Unknown Concentration
Bradford Assay
1.2
y = 0.96x + 0.06
R2 = 0.973
1
Absorbance 595 nm
0.8
0.6
0.4
0.2
0
0 0.2 0.4 0.6 0.8 1 1.2
Protein Concentration (mg/mL)
Today’s Activity
• Measure the absorbance of a standard
curve and an unknown protein sample
• Plot the standard curve and determine the
unknown protein concentration.