Application Guide

Cubetas de cuarzo para cuantificación de proteínas: A280, Bradford y BCA

La cuantificación de proteínas se realiza en dos longitudes de onda muy distintas, y ese solo hecho determina la cubeta necesaria. La absorbancia UV directa a 280 nm requiere cuarzo: ni el plástico ni el vidrio óptico transmiten ahí. Bradford y BCA leen en el visible (562–595 nm), donde también sirven vidrio y plástico desechable. Esta guía cubre ambos casos.

1. Por qué A280 requiere cuarzo

Proteins absorb UV at 280 nm through their aromatic residues — tryptophan and tyrosine primarily, with a smaller contribution from phenylalanine and disulfide bonds. Measuring there directly is fast, reagent-free, and the sample is recoverable, which matters when the protein is the product and not just the analyte.

But 280 nm is deep UV. Disposable polystyrene cuvettes cut off around 340 nm and optical glass around 340 nm as well — both are opaque at 280 nm. Only fused silica transmits there. JGS1 synthetic fused silica covers 200–2,500 nm, so a quartz cell handles A280 and every visible assay on the same instrument.

2. UV directo (A280) frente a ensayos colorimétricos

Method	Wavelength	Cuvette material	Sample recoverable?
A280 direct UV	280 nm	Quartz (required)	Yes
Bradford	595 nm	Quartz, glass or plastic	No
BCA	562 nm	Quartz, glass or plastic	No
Lowry	650–750 nm	Quartz, glass or plastic	No

3. Elección del paso óptico

Beer-Lambert linearity holds roughly between 0.1 and 1.0 absorbance units. Outside that window your numbers drift, and no amount of cuvette quality fixes it.

Dilute samples (<0.1 AU at 10 mm) — stay at the standard 10 mm path rather than concentrating.
Concentrated samples (>1.0 AU at 10 mm) — switch to a short path cell (1–5 mm) instead of diluting. This keeps the reading in range and avoids pipetting error.
Unknown concentration — measure at 10 mm first, then pick the path length that lands you between 0.1 and 1.0 AU.

4. Volumen: cubetas micro y sub-micro

A standard 10 mm macro cell needs roughly 3.5 mL; a semi-micro around 1–1.5 mL. Purified protein rarely comes in those quantities. Micro and sub-micro cells with a masked (black-walled) chamber bring the working volume down to 50–500 µL while keeping the 10 mm path the instrument expects, because the light beam is narrowed rather than the path shortened.

If your sample volume is the binding constraint rather than concentration, this is the first specification to change.

5. Cubetas recomendadas

A280 work — JGS1 quartz macro cells, 10 mm. Transmission to 200 nm means 280 nm sits in a flat, low-noise part of the curve.
Limited sample — micro and semi-micro quartz cells (black-walled versions available).
Very concentrated — short path length cells, 1–5 mm.
Bradford / BCA only — quartz still preferred for reuse and solvent resistance, but optical glass is an acceptable economy option.

6. Notas prácticas

Blank with the same buffer the protein is in — buffer absorbance is not zero at 280 nm.
Detergents (SDS, Triton) absorb strongly below 280 nm and will distort A280. Use a detergent-compatible colorimetric assay instead.
Rinse protein residue promptly; dried protein film is the most common cause of a drifting baseline. See the cleaning protocol.
Verify your cell at the wavelength you actually use — a cell certified at 600 nm tells you nothing about 280 nm.

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