Question 1

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 2

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 3

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 4

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 5

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 6

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 optimize your downstream process.

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