How the Direct Detect® spectrometer uses FTIR Technology for protein quantitation

IR spectroscopy is one of the most well established techniques for the analysis of protein structure. IR spectroscopy exploits the fact that molecules absorb radiation at specific frequencies characteristic of their structure. By measuring amide bonds in protein chains, the Direct Detect spectrometer accurately quantifies an intrinsic component of every protein without relying on amino acid composition, dye binding or redox potential. Analyze components of complex mixtures--proteins, lipids, carbohydrates, and nucleic acids have separable IR spectra.

Advantages of FTIR Quantitation:

Enhanced Protein quantitation using FTIR technology
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.

Accuracy of FTIR spectroscopy is independent of detergents and reducing agents

IR-based protein quantitation using the Direct Detect system involves measuring the intensity of the Amide I peak in the protein’s IR spectrum and subtracting the signal contributed by buffer alone in that region. As a result, IR-based quantitation retains accuracy and precision in the presence of SDS or reducing agents such as DTT.

FTIR compared to colorimetric assays

Unlike colorimetric Bradford and MicroBCA assays, which indirectly detect the products of secondary reactions, the IR signal of a protein does not depend on reaction kinetics. Because the IR signal is independent of the time point at which the measurement is made, and because this method is compatible with detergents and reducing agents, IR-based quantitation is a more convenient, flexible, universal approach to measuring protein levels in complex mixtures, compared to the Bradford and BCA colorimetric assays. Also, because IR-based quantitation only requires a standard curve to be generated once (instead of for every experiment), this method is often faster than quantitation using colorimetric assays.

Spectroscopic Principles of Direct Detect® Quantitation

Infrared (IR) spectroscopy exploits the fact that molecules absorb specific frequencies characteristic of their structure. To form a protein or a peptide, amino acids are covalently linked via amide (peptide) bonds. Amide bonds absorb electromagnetic radiation in multiple regions of the mid-IR spectrum, including the strong band at 1600-1690 cm-1. In order to determine protein and peptide concentration, the Direct Detect Spectrometer measures the intensity (peak height) of the Amide I band, which is assigned to C=O stretching vibration of the peptide bond (about 80%) with a minor contribution from C-N stretching vibration (about 20%).

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