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

gure, the highlighted plot is Plate with PathCheck. The data came from the group Plate Standard, the x variable is concentration, and the y variable is MeanValue (i.e. the mean pathlength-corrected absorbance value).


The resulting graph ( Figure 8 ) shows the three plots (plate values with and without PathCheck and the cuvette values). The plot of raw plate absorbance values without PathCheck (bottom line) is irregular because the volumes in the wells (and therefore the pathlengths in the wells) were not uniform. The plot of plate values with PathCheck is linear through the highest concentration (6 mg/ mL). The corrected plate values are superimposable on the cuvette values up through approximately 3 mg/ml, above which the cuvette plot begins to deviate from linearity. The corrected plate plot extends above the cuvette plot because the values are calculated values and their raw absorbance values (bottom plot) are still well within the linear range of the curve.


Example 2: Using PathCheck to evaluate column fractions during purification of antibody-enzyme conjugate on a gel filtration column
Fractions were collected during purification of an antibody-enzyme conjugate on a Pharmacia S-100 gel filtration column. The fractions in the void volume were discarded and fractions 21 through 61 were each read at 280 nm in a Costar UV plate and also in a standard quartz cuvette for comparison. Fractions 24 through 29 were too concentrated and therefore were diluted 1 to 5 to bring them into the linear range of absorbency.

Initial setup of the Group Table
There are two ways in SOFTmax PRO to set up a Group Table for samples (e.g. column fractions) that have dilution factors, but are not going to be quantitated against a standard curve:

1. Assign the Unknowns(dilution) format to the group. SO
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