( INTRODUCTION ...)PathCheck Applied to Measurement of Protein Solutions in the SpectraMax Plus Microplate Spectrophotometer (MaxLine Application Note #26)

INTRODUCTION
Protein concentration is commonly estimated by measuring the absorbance at 280 nm and calculating the concentration using the extinction coefficient of the protein. The procedure is feasible because extinction coefficients are typically obtained using standard cuvettes with a fixed 1 cm pathlength. Extinction-based protein assays can also be done in microplates, but they have not been widely-used because of the inconvenience of compensating for variable pathlengths of samples in microplate wells. The introduction of the Molecular Devices SPEC-TRAmax PLUS microplate spectrophotometer marks the first time that photometric measurements made in a microplate format can be automatically normalized such that the resultant values are identical to those obtained with the corresponding solutions in a standard 1 cm pathlength cuvette. The following application note outlines the use of the pathlength correction feature Pathcheck, compares absorbance results obtained in cuvettes and microplate, and gives an example where Pathcheck is applied to column chromatographic fractions and the elution profile displayed.

MATERIALS
For details of the principles of pathlength correction, refer to MAXline application note #25, Optical density measurements automatically normalized to a 1 cm pathlength in the SPECTRAmax PLUS microplate spectrophotometer.

1. SPECTRAmax PLUS microplate spectrophotometer

2. UV-transparent microplates; e.g.:
SPECTRAplate Quartz, Molecular Devices Corp. (catalog # R8024)
SPECTRAplate Quartz half-area microplate, Molecular Devices Corp., (catalog # R8028)
Quartz microtest 8-well strip, Hellma (catalog # 730.010QS); use with a frame for 8-well strips, E & K Scientific Products (catalog # 564101). Note: the 8-well strip frame sold by Evergreen Scientific does no
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