Protein Quantitation with Direct Detect® Spectrometer

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Direct Detect® FTIR quantitation outperforms Bradford and BCA protein assays in samples containing detergents and reducing agents.

The Direct Detect® provides accurate and precise results, even in presence of detergent (SDS) and reducing agent (DTT). Using the Direct Detect® IR-based quantitation system, calculated concentrations for all the BSA samples matched the prediluted standard (Thermo BSA Standard). In comparison, the Coomassie Plus (Bradford) assay provided calculated concentrations that differed greatly in the presence of 1% SDS, and the MicroBCA assay could not provide data in the presence of 50 mM DTT.

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Compare Direct Detect® FTIR quantitation with Bradford and BCA protein assays in samples.

Quantitation Approach	Assay Range	Sample Volume (tube/plate)	Amount Protein Required to Measure 1 mg/mL	Compatible
Direct Detect® Spectrometer	250-5,000 μg / mL	2 μL	6 μg	Detergents Reducing agents SDS Chelators
Bradford Assay	100-1,500 μg / mL	35 μL / 7 μL	21 μg	Most reducing agents Chelators
BCA Assay	20-2,000 µg / mL	50 µL / 25 µL	30 μg	Detergents
Micro BCA™ Assay	2-40 µg / mL	500 µL / 150 µL	450 μg	Detergents
Lowry Assay	10-1,500 μg / mL	200 μL / 40 μL	120 μg	SDS

How FTIR Protein Quantitation Works in Complex Samples:

Protein quantitation in presence of lipids within a complex cell lysate is possible because the most intense regions of lipid absorbance are spectrally distinct from the protein’s Amide I and Amide II signals.