Poster Presentation 6th Australian Health and Medical Research Congress 2012

Improving Protein Detection Using IR-Based Quantitation (#449)

Chris Utzat 1 , Ivona Strug 1 , Ryan Amara 1 , Nuno Goncalves 1 , Joe Lento 1 , Tim Nadler 1 , Mark Kraschnefski 2
  1. EMD Millipore Corporation, Billerica, Massachusetts, United States of America
  2. Merck Millipore, Kilsyth, VIC, Australia

The Direct Detect™ IR-based quantification system represents an innovation in biomolecule quantitation. By directly measuring amide bonds in protein chains, the Direct Detect™ system accurately determines an intrinsic component of every protein without relying on amino acid composition.

Infrared (IR) spectroscopy is one of the oldest and most well established experimental techniques for the analysis of polypeptides and protein structure. It is convenient, requires minimal sample preparation and can be used under a wide variety of conditions. IR spectroscopy exploits the fact that molecules absorb specific frequencies that are characteristic of their structure. Protein primary structure is formed by a long chain of amide bonds that are represented by nine IR absorbance areas. In order to determine protein concentration Direct Detect™ IR-based technology utilizes Amide I band, which is assigned to the C=O stretching vibration within the peptide bond.

Within minutes and without any bio- or immuno-chemical staining, protein concentration in an extremely wide range from about 0.2 mg/mL to >20 mg/mL can be determined directly from the undiluted solution. In addition, amide bond quantitation is not subject to interference from many common buffer components such as detergents, reducing agents and chelators. This allows for direct quantitation of proteins without the need to run Bradford, BCA or other colorimetric assays.