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

each time, to avoid over-writing the pre-read data.

If you use Costar UV plates, you may or may not want to use stored pre-read values, depending on the precision you require. At 280 nm, the average absorbance of Costar wells containing water is approximately .050 + .002 OD. Thus, samples having raw absorbance values of 1 OD would be subject to less than 1% potential error due plate background variation. However, the lower the absorbance in the wells, the greater the maximum potential error due to plate variability.


RESULTS
Example 1: Demonstration of the utility of PathCheck using a protein standard curve
To illustrate the performance of the instrument, a protein standard curve was prepared. Bovine serum albumin (BSA) was dissolved in phosphate-buffered saline (PBS), pH 7.4, filter-sterilized and diluted to 6, 5, 4, 3, 2, 1, 0.5 and 0.25 mg/ mL. Absorbance measurements made in a microplate were compared with measurements made on the same solutions in a 1 cm cuvette.

SOFTmax PRO was launched and a Group entitled Plate Standard was created and given a Standards format in the Template Editor of a Plate section. The standards were assigned to wells as illustrated in Figure 3. Standard 01 was PBS with no added protein (0 mg/mL). An identical template was set up in the Template Editor of a Cuvette Set, except the group was named Cuvette Standard.


The instrument settings and data reduction for the Plate section were set up as shown in Figures 1 and 2 above. The CuvetteSet section was set up for an endpoint read at 280 nm.

A cuvette containing PBS was placed in the cuvette port to serve as the cuvette reference for PathCheck. Pre-read measurements were made with 200 L water in each well of a Costar UV plate, afte
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