( r which the plate was emptied and blott...)PathCheck Applied to Measurement of Protein Solutions in the SpectraMax Plus Microplate Spectrophotometer (MaxLine Application Note #26)

r which the plate was emptied and blotted dry. Aliquots of the protein standard solutions were transferred into their designated wells using disposable transfer pipets. The volumes in each well were deliberately varied in order to demonstrate the utility of the PathCheck feature. The plate was placed into the drawer of the microplate reader and read.

In the Cuvette Set, a reference read was taken on the PBS-containing cuvette in the cuvette port. Then, one by one, each of the protein standard solutions was read in the cuvette.

The Group Table associated with the Plate section is shown in Figure 4.

All columns except the far right column in the Group Table were created automatically by SOFTmax PRO when the Plate Standard group was created in the template. The numbers in the Values column are absorbance values corrected to a 1.0 cm pathlength. The numbers in the far right column titled Raw Values are raw absorbance values (pre-read subtracted) before pathlength correction. These values were obtained by back-calculation using the custom formula shown in the Calculation dialog box for that column ( Figure 5). Basically, the Raw Values were calculated by multiplying the corrected values by their corresponding pathlengths and taking the average of the replicates.

The Group Table (Cuvette Standard) displays the data from the absorbance readings in the cuvette ( Figure 6). The column Values contains the absorbance values at 280 nm.

Next, the standard curves from the plate and cuvette were plotted in a single graph. A Graph section was created, then the Plots dialog box was used to create three plots by selecting the corresponding Group tables from the Groups pop-up menu, then specifying the x and y variables for each plot ( Figure 7). In the example shown in the fi
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