What you are given
- A280: 0.667
- 0.1% extinction (A280 of 1 mg/mL): 0.667
Formula: c (mg/mL) = A280 / (E0.1% x path)
Protein
How do you calculate protein concentration from A280?
concentration in mg/mL = A280 / (0.1% extinction coefficient x path length). The extinction coefficient depends on the protein, since it comes from its tryptophan, tyrosine, and cystine content.
Worked example
Every number below is computed by the calculator, not typed in
The working
- Solve for concentration:
conc = A280 / (E0.1% · path) - Substitute:
conc = 0.667 / (0.667 · 1 cm)=1 mg/mL
Your protein is 1 mg/mL.
What each symbol means
- c: Protein concentration (mg/mL)
- A₂₈₀: Absorbance at 280 nm
- ε0.1%: 0.1% extinction coefficient (A280 of 1 mg/mL, 1 cm) (mL/(mg cm))
- ell: Path length (cm)
A printable one-page version is on the formula sheet.
Where this usually goes wrong
- Using a generic extinction coefficient for an unknown protein. It can be off by several fold.
- Not subtracting scatter. Turbidity inflates A280.
- Reading a protein with no aromatic residues, which barely absorbs at 280 at all.
Try it yourself
Two problems, with the working revealed
Problem 1 Protein by A280
Work out the protein concentration.
- A280: 0.4
- 0.1% extinction (A280 of 1 mg/mL): 0.667
Show the answer and the working
Your protein is 0.5997 mg/mL.
- Solve for concentration:
conc = A280 / (E0.1% · path) - Substitute:
conc = 0.4 / (0.667 · 1 cm)=0.5997 mg/mL
Open this in the calculator to change the numbers.
Problem 2 Protein by A280
Work out the protein concentration.
- A280: 0.75
- 0.1% extinction (A280 of 1 mg/mL): 0.667
Show the answer and the working
Your protein is 1.124 mg/mL.
- Solve for concentration:
conc = A280 / (E0.1% · path) - Substitute:
conc = 0.75 / (0.667 · 1 cm)=1.124 mg/mL
Open this in the calculator to change the numbers.
Do it with your own numbers.
The Protein by A280 calculator is free, shows every step, flags the hazards, and works offline once loaded. No account, no signup.
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