Question 1

What is an A280 protein concentration calculator?

Accepted Answer

A280 absorbance is the UV absorbance at 280 nm, where tryptophan, tyrosine and cystine absorb; concentration = A280 / (epsilon x b), where epsilon is the extinction coefficient (IgG 1.4 mg/mL, BSA 0.667 mg/mL at 1 cm path length) and b is the pathlength in cm. An A280 protein concentration calculator applies this Beer-Lambert equation, looks up or accepts a custom extinction coefficient, corrects for the NanoDrop pedestal path length (0.1 cm vs 1 cm), applies the Warburg-Christian A260 nucleic-acid correction when needed, multiplies by the dilution factor, and returns the result in mg/mL with SD/CV across replicates.

Question 2

How accurate is A280 vs BCA?

Accepted Answer

A280 is accurate to approximately plus/minus 5 percent for a pure protein with a known extinction coefficient and no nucleic acid contamination (A260/A280 < 0.6). BCA tolerates detergents and most buffer salts but is sensitive to reducing agents (DTT, beta-mercaptoethanol, TCEP) and metal chelators (EDTA), and typically gives plus/minus 10 percent CV when a fresh BSA standard curve is run per batch. In short: A280 wins on precision for a purified reagent-free sample, BCA wins on robustness in detergent-containing lysates.

Question 3

Which protein concentration assay should I choose (BCA, Bradford, A280)?

Accepted Answer

Choose A280 for a purified, reagent-free protein when you know the extinction coefficient. It reads in seconds from ~0.1 to 3 mg/mL, but is blocked by nucleic acid contamination at 260 nm and underestimates proteins low in Trp/Tyr/cystine. Choose BCA (bicinchoninic acid) for detergent-containing lysates: linear range ~0.02 to 2 mg/mL, 30 min at 37 C, reads at 562 nm, tolerates SDS and Triton X-100 but is blocked by reducing agents (DTT, BME, TCEP) and metal chelators (EDTA). Choose Bradford (Coomassie G-250) for a fast (~5 min) read on small volumes without detergents: linear range ~0.1 to 1.5 mg/mL, reads at 595 nm, high protein-to-protein variability. In short: A280 for pure, BCA for detergent-heavy, Bradford for fast and clean.

Question 4

What formula converts A280 to protein concentration in mg/mL?

Accepted Answer

Use the Beer-Lambert law: C (mg/mL) = A280 / (epsilon x l), where epsilon is the extinction coefficient at 1 mg/mL (often written epsilon_0.1%, dimensionless) and l is the path length in cm. Typical values at 1 mg/mL: IgG ~1.4, BSA ~0.66, generic mAb ~1.5, lysozyme ~2.65. Path length is 1 cm for a standard cuvette and 0.1 cm on a NanoDrop pedestal. Multiply the result by any dilution factor. For an exact epsilon from an amino acid sequence, use ExPASy ProtParam or the Protein MW and Extinction Coefficient Calculator at https://bioprocesstools.com/protein-mw-calculator/. If the sample has visible A260 contamination, apply the Warburg-Christian correction: C (mg/mL) = 1.55 x A280 - 0.76 x A260.

Question 5

How to calculate protein concentration from BCA assay?

Accepted Answer

Run a BSA standard curve (0, 25, 125, 250, 500, 1000, 2000 ug/mL) alongside your samples, incubate with BCA working reagent for 30 min at 37 C and read absorbance at 562 nm. Blank-subtract every reading, fit a linear regression of corrected A562 versus BSA concentration (y = m*x + b), then compute unknown concentration = (sample_A562 - b) / m and multiply by the dilution factor. Paste your standards and sample readings into this Protein Concentration Calculator (BCA tab) at https://bioprocesstools.com/protein-concentration-calculator/ and it returns the concentration in ug/mL with R2 and standard curve chart instantly.

Question 6

How do I convert A280 absorbance to protein concentration?

Accepted Answer

Use the Beer-Lambert law: concentration (mg/mL) = A280 / (epsilon_0.1% x path length), where epsilon_0.1% is the 0.1 percent extinction coefficient at 280 nm (IgG ~1.4, BSA ~0.66, generic mAb ~1.5) and path length is 1 cm for a cuvette or 0.1 cm on a NanoDrop. Divide the A280 reading by the extinction coefficient and correct for path length, then multiply by the dilution factor. The Direct A280 tab of this Protein Concentration Calculator at https://bioprocesstools.com/protein-concentration-calculator/ does the conversion instantly, including NanoDrop path length, the Warburg-Christian nucleic-acid correction and multi-replicate SD/CV.

Question 7

What is the difference between BCA and Bradford protein assays?

Accepted Answer

The BCA (bicinchoninic acid) assay detects Cu+ ions produced by protein-mediated reduction of Cu2+ in alkaline conditions, reading at 562 nm. It is compatible with most detergents and denaturants. The Bradford assay uses Coomassie Brilliant Blue G-250 dye binding, reading at 595 nm. Bradford is faster (5 min vs 30 min incubation) but is incompatible with detergents like SDS and Triton X-100. BCA has a broader linear range (20-2000 ug/mL) compared to Bradford (125-1000 ug/mL for standard, 1-25 ug/mL for micro).

Question 8

How do I calculate protein concentration from A280?

Accepted Answer

Protein concentration from A280 uses Beer-Lambert law: A = epsilon x c x l, where A is absorbance at 280 nm, epsilon is the molar extinction coefficient (M-1 cm-1), c is molar concentration, and l is path length (typically 1 cm). Rearranging: c (mg/mL) = A280 / (epsilon_0.1% x l), where epsilon_0.1% is the extinction coefficient for a 1 mg/mL solution (= epsilon / MW x 1000). This method requires a pure protein sample and known extinction coefficient.

Question 9

Why does my BCA standard curve not pass through zero?

Accepted Answer

BCA standard curves rarely pass exactly through zero because the BCA reagent itself has a slight background absorbance, and trace metals or buffer components can cause minor Cu2+ reduction. This is normal. Always include a blank (0 ug/mL standard) and subtract its absorbance from all readings before fitting. A y-intercept up to about 0.05 AU is typical. If the intercept is much higher, check for interfering substances (reducing sugars, lipids, EDTA, DTT) in your sample buffer.

Question 10

What protein standard should I use for BCA or Bradford assays?

Accepted Answer

BSA (bovine serum albumin) is the most common standard because it is inexpensive, stable, and gives reproducible results. However, protein-to-protein variation means your target protein may respond differently. IgG standards are recommended when quantifying antibodies, as immunoglobulins bind Coomassie dye differently from albumins. For the most accurate results, use a purified batch of your target protein as the standard. Report concentrations as 'BSA equivalents' or 'IgG equivalents' when using a surrogate standard.

Question 11

Can I use A280 to measure protein in crude lysate?

Accepted Answer

A280 is not recommended for crude lysates because nucleic acids absorb strongly at 280 nm (and even more at 260 nm), causing significant overestimation. The A260/A280 ratio can partially correct for nucleic acid contamination: corrected protein (mg/mL) = 1.55 x A280 - 0.76 x A260 (Warburg-Christian method). For crude samples, BCA or Bradford assays are more appropriate. A280 is best suited for purified protein solutions where nucleic acid contamination is minimal (A260/A280 < 0.6).

Question 12

How do I calculate purification yield from concentration data?

Accepted Answer

Purification yield (%) = (total protein after step / total protein before step) x 100. Total protein = concentration (mg/mL) x volume (mL). For example, if you start with 50 mL at 10 mg/mL (500 mg total) and after Protein A chromatography you have 5 mL at 80 mg/mL (400 mg total), the step yield is 400/500 x 100 = 80%. Track yield at every purification step (capture, intermediate purification, polishing) to identify steps with excessive losses and optimise your downstream process.

Question 13

How does the A280 protein concentration calculator work?

Accepted Answer

Our A280 protein concentration calculator applies the Beer-Lambert law: A280 = epsilon_0.1% x c (mg/mL) x l (cm), where epsilon_0.1% is the 0.1% extinction coefficient at 280 nm and l is the cuvette path length (1 cm by default, 0.1 cm for NanoDrop). The calculator includes a built-in extinction coefficient library (IgG 1.4, BSA 0.66, lysozyme 2.65, transferrin 1.43, generic mAb 1.5) and accepts a custom value computed from your sequence via ExPASy ProtParam. It also supports the Warburg-Christian nucleic-acid correction (c_mg/mL = 1.55 x A280 - 0.76 x A260) for samples with low A260/A280 contamination. Path length, dilution factor, and number of replicates are all configurable. Output is in mg/mL with automatic SD/CV across replicates.

Method	Principle	Wavelength	Linear range	Time	Detergent / reducing-agent compatibility	Best use
A280	Intrinsic Trp/Tyr/cystine UV absorbance (no reagents)	280 nm	~0.1–3 mg/mL (protein-dependent)	Seconds	Reagent-free; sensitive to nucleic-acid contamination at 280 nm	Relatively pure protein with a known extinction coefficient
Bradford	Coomassie G-250 dye binding	595 nm	~1–20 µg	~10 min	Incompatible with detergents; high protein-to-protein variability	Fast reads on detergent-free samples
BCA	Cu²⁺ reduction + bicinchoninic acid chelation	562 nm	~20–2000 µg	30 min at 37 °C	Detergent-compatible, but interfered by reducing agents/chelators; lower protein-to-protein variability	Wide range in detergent-containing lysates

A280 Protein Concentration Calculator: Extinction Coefficient Tool

About the A280, BCA and Bradford Protein Concentration Calculator

A280 vs Bradford vs BCA: which protein assay to use

Method references & further reading

Further reading

A280 Protein Concentration Calculator: Extinction Coefficient Tool

Protein Aggregation Guide

Buffer Preparation Guide

Protein A Resin Lifetime Study

Biopharmaceutical COGS Analysis

About the A280, BCA and Bradford Protein Concentration Calculator

A280 vs Bradford vs BCA: which protein assay to use

Method references & further reading

Further reading