Quarzküvetten zur Proteinquantifizierung: A280, Bradford und BCA

Die Proteinquantifizierung arbeitet bei zwei sehr unterschiedlichen Wellenlängen, und allein diese Tatsache bestimmt die benötigte Küvette. Direkte UV-Absorption bei 280 nm erfordert Quarz – weder Kunststoff noch optisches Glas transmitieren dort. Bradford und BCA messen im Sichtbaren (562–595 nm), wo auch Glas und Einwegkunststoff funktionieren. Diese Anleitung behandelt beide Fälle.

1. Warum A280 Quarz benötigt

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. Direktes UV (A280) vs. kolorimetrische Assays

3. Wahl der Schichtdicke

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: Mikro- und Submikroküvetten

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. Empfohlene Küvetten

• 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. Praktische Hinweise

• 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.

Related: Cleaning Protocol · UV-Vis Troubleshooting · Solvent Compatibility · Path Length Guide · Path Length Calculator · Cuvette Care Guide