Protein Concentration Calculator (A280)
Determine the concentration of a purified protein sample based on its absorbance at 280 nm, molar extinction coefficient, and molecular weight.
Protein Spectrophotometry
Specify absorbance, extinction coefficient, path length, and molecular weight.
Protein Concentration
* Equal to 0.271 g/L.
Linear Dynamic Range
Ensure A280 is within 0.1 to 1.5 AU. Dilute samples if A280 exceeds 1.5 to maintain Beer-Lambert linearity.
Standard Protein Extinction Coefficients & Mass Absorbance Reference
| Standard Protein / Antibody | MW (kDa) | A280 for 0.1% (1 mg/mL) | Molar Extinction ε (M⁻¹ cm⁻¹) |
|---|---|---|---|
| Bovine Serum Albumin (BSA) | 66.4 kDa | 0.667 AU | 43,824 M⁻¹ cm⁻¹ |
| Human Immunoglobulin G (IgG) | 150.0 kDa | 1.370 AU | 210,000 M⁻¹ cm⁻¹ |
| Lysozyme (Hen Egg White) | 14.3 kDa | 2.640 AU | 37,750 M⁻¹ cm⁻¹ |
| Ovalbumin | 42.7 kDa | 0.735 AU | 31,390 M⁻¹ cm⁻¹ |
Calculation Methodology & Equations
Beer-Lambert Molarity
Calculates molar concentration where A₂₈₀ is absorbance, ε is molar extinction coefficient (M⁻¹ cm⁻¹), and L is cuvette pathlength (cm).
Mass Concentration Conversion
Converts molar concentration to mass concentration using molecular weight in kDa (g/mmol).
Frequently Asked Questions
Why do proteins absorb UV light at 280 nm? ▼
Protein UV absorbance at 280 nm is primarily driven by the aromatic side chains of tryptophan (Trp) and tyrosine (Tyr), and to a minor extent disulfide bonds (cystine).
How is molar extinction coefficient (ε) calculated for a protein sequence? ▼
ε280 (M⁻¹ cm⁻¹) = (N_Trp × 5,500) + (N_Tyr × 1,490) + (N_Cystine × 125), where N is the number of respective residues in the folded or unfolded chain.
When should colorimetric assays (BCA, Bradford) be used instead of A280? ▼
Colorimetric assays should be used if the protein lacks tryptophan/tyrosine residues, if buffer components absorb at 280 nm (e.g. nucleic acids, Triton X-100), or if crude cell lysates are being measured.
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