Protein quantitation in complex samples

Direct Detect FTIR Quantitation outperforms Bradford and BCA Protein Assays in Samples Containing Detergents and Reducing Agents

Direct Detect outperforms Bradford assay, BCA protein assay, and lowry assay for protein quantitation
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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.


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 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.