Direct Detect Spectrometer

BACK TO PROTEIN ANALYSIS
ROOM N232, ANDERSON STUART BUILDING (F13), ROOM 320, BLACKBURN BUILDING (D06)

The Direct Detect infrared-based quantification system is an innovative combination of software-controlled instrumentation and Merck Millipore Corporation’s advanced membrane technology, optimised for the detection and quantification of proteins. Biomolecules are applied directly to a card-based hydrophilic polytetrafluoroethylene (PTFE) membrane that is transparent in most of the infrared spectral region.

The Direct Detect system measures amide bonds in protein chains, accurately quantifying an intrinsic component of every protein without relying on amino acid composition, dye binding properties, or redox potential.

Protein concentrations from 0.25 to 5 mg/ml can accurately be determined from a minimal sample volume (2 μl) without bio- or immuno-chemical staining. Sample analysis takes only minutes and in most cases can be performed directly from the buffered or native solution.

The Direct Detect system is compatible with most commonly used buffers, detergents, and reducing agents, however, use of buffers which contain amides or amine functional groups may affect quantification. Examples of buffers and respective concentrations which interfere with the spectral region used for protein quantification are listed below.

Bicarbonate (divalent) > 25mM, EDTA > 10mM, Guanidine hydrochloride > 8mM, Guanidine isothyocyanate > 4mM, HEPES buffer (pH7.5) > 25mM, Sodium bicarbonate > 50mM, Sodium hydroxide > 100mM, Tris > 50mM, Urea > 2mM.

A secondary analysis method should be considered when quantifying in the presence of these buffers.

Buffers containing more than 5% glycerol, SDS, and/or Tween20 surfactant should be avoided due to the excessive time required to achieve dryness.

Calibration with the buffer used for protein preparation is recommended.

GUIDELINES
The Direct Detect Spectrometers are located in room N232, Anderson Stuart building (F13) and room 320, Blackburn Building (D06). Online booking system is available. You need to be trained on how to use the system by the Molecular Biology Officer before you can use it independently. Please cover the card holder after use. Thanks.