PathCheck applied to measurement of protein solutions in the SPECTRAmax ® PLUS microplate spectrophotometer INTRODUCTION
Protein concentration is commonly estimated by measuring the absorbance at 280 nm and calculating the concentration using the extinction coefficient of the pro-tein. The procedure is feasible becau extinction coefficients are typically obtained using standard cuvettes with a fixed 1 cm pathlength. Extinction-bad protein assays can also be done in microplates, but they have not been widely-ud becau 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 photomet-ric measurements made in a microplate format can be automatically normalized such that the resultant values are identical to tho obtained with the correspond-ing solutions in a standard 1 cm pathlength cuvette. The following application note outlines the u 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”.
1SPECTRAmax PLUS microplate spectrophotometer
2UV -transparent microplates; e.g.:aah怎么读
•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); u with a frame
for 8-well strips, E & K Scientific Products (catalog # 564101). Note: the 8-well
strip frame sold by Evergreen Scientific does not fit properly.
•Disposable UV Plate, Corning Costar Corp. (catalog # 3635)
•UV -MAX , (Polyfiltronics, Inc., catalog # VO32SCXP8)
Application Note 26
3Pipettor and tips or transfer pipets suitable for u with microplates.
4Samples (100-300 µL each)
5Quartz cuvette
Set up to make pre-read measurements
Step 1Launch SOFTmax PRO®, then open a Plate ction or, if necessary, cre-ate a new Plate ction.
Step 2Set up the Instrument Settings dialog box as shown in Figure 1. In the Instrument Settings dialog box, lect to perform an endpoint read at
280 nm. Select PathCheck, (Pre-read plate will automatically be
checked) and lect Cuvette Reference. (Alternatively, you can u the
factory-installed Water Constant as a shortcut, though it is potentially
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less accurate.)
Figure 1: The Instrument Settings dialog box, t up to measure absorbance at 280 nm
with PathCheck
Step 3U the Template Editor to create a template showing where standards, blanks, and unknowns will be located on the microplate.
Step 4Set up the Reduction dialog box as shown in Figure 2. Set the wave-length combination to L1 and the data mode to Absorbance. Make sure
that ‘U pre-read plate’ and ‘Apply Pathcheck’ are lected.
Figure 2: The Reduction dialog box t up to apply PathCheck
METHOD Making pre-read measurements
The purpo of the pre-read is to subtract the background absorbance of the plate.
In contrast, blanks (plate, sample or group) are designated in the template and are
subtracted after pathlength correction. In some cas, you may choo to u
stored pre-read values (See below).
Step 1Select a clean microplate and pipet distilled water into the wells in
approximately the same volumes as the intended samples. Accurate
pipetting is not necessary.
Step 2Put the plate into the drawer of the SPECTRAmax PLUS, then click the
Read button in SOFTmax PRO’s toolbar to make the pre-read measure-
ments.
Step 3Remove the plate, pour out the water and blot the plate dry.
Making pathlength-corrected absorbance measurements
Note: If you have elected to u the pre-programmed Water Constant, you may skip Step 1.
Step 1Place a clean quartz cuvette containing your sample buffer or distilled
water into the SPECTRAmax PLUS cuvette port.
Step 2Transfer aliquots of your samples and blanks into their designated wells
in the microplate. Accurate pipetting is not necessary, though the vol-
umes should between 100 µL and 300 µL for best pathlength-corrected
results.
Step 3Put the plate into the SPECTRAmax PLUS drawer, then click the Read
button in SOFTmax PRO’s toolbar to make the absorbance measure-
ments. (The cuvette will be read in the near-infrared at the same time
the plate is read, and its absorbance values ud in the PathCheck calcu-
lations.)
executionTo avoid evaporation error, make the readings within a few minutes of
putting the samples in the microplate. If the readings must be delayed,
cover the plate with an adhesive al. Remove the al immediately
prior to reading the plate.
Storing and re-using pre-read data
Quartz plates are so uniform that it is not necessary to subtract the plate back-
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ground on a well-by-well basis. Instead, you can pre-read one plate, then store the
pre-read data in a SOFTmax PRO file for subquent re-u, being careful to save
the data under a different file name each time, to avoid over-writing the pre-read
data.
If you u Costar UV plates, you may or may not want to u 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 rum 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 mea-
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surements 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 ction. The
standards were assigned to wells as illustrated in Figure 3. “Standard 01” was
estimatedPBS with no added protein (0 mg/mL). An identical template was t up in the
Template Editor of a Cuvette Set, except the group was named “Cuvette
Standard”.
Figure 3:
The Template Editor for the Plate ction
The instrument ttings and data reduction for the Plate ction were t up as
shown in Figures 1 and 2 above. The CuvetteSet ction was t up for an
endpoint read at 280 nm.
A cuvette containing PBS was placed in the cuvette port to rve as the cuvette
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reference for PathCheck. Pre-read measurements were made with 200 µL water
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in each well of a Costar UV
plate, after 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 ction is shown in Figure 4. Figure 4:
研究生考试时间安排The Group Table for the Plate ction
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. The 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.
Figure 5:
cathay
The Calculation dialog box for the “Raw Values” column
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.
Figure 6: The Group Table for the Cuvette Set
Next, the standard curves from the plate and cuvette were plotted in a single graph. A Graph ction was created, then the Plots dialog box was ud to create three plots by lecting 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 figure, the highlighted plot is “Plate with PathCheck”. The da
ta 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).
Figure 7: The Plots dialog box associated with the Graph Section
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 becau 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
