User manual

Section 3- Applications
Note: If selecting a previously saved workbook, all concentration calculations for newly measured
samples will be based upon the standard absorbance values saved in the workbook. Each
workbook will archive only one standard curve.
Making BCA Assay Measurements
• Refer to the manufacturers’ guidelines and recommendations for sample preparation.
• The Zero Reference standard is a solution of the same buffer/dye reagent composition as the other
standards and samples but without any protein added.
• Prepare both standards and unknowns in the same manner. Use a diluent of the same pH and ionic
strength for all blanks, standards and unknown samples.
• Standards diluted from a stock must cover the expected range of the unknown samples. Sample protein
concentrations are not extrapolated beyond measured standard concentrations.
Procedure
1. Select the Protein BCA application from the main menu. If the wavelength verification window appears,
ensure the arm is down and click OK.
2. Enter the values for each standard concentration in the right pane table. The software allows for the
reference and up to 7 additional standards. The Reference and/or standards can be measured in replicates.
Note: The minimum requirement for standard curve generation is the measurement of two standards
or the measurement of the zero reference and at least one standard. It is recommended that
additional standards be included as necessary to cover the expected assay concentration
range.
3. Select Add to report to automatically include all measurements in the current report. The default setting is
for all samples to be added to reports. The Add to report checkbox must be selected prior to a
measurement to save the sample data to a workbook.
- Select the file drop-down option Use current settings as default as a convenient way to limit set-up time
for each new workbook.
4. Select Overlay spectra to display multiple spectra at a time.
5. Establish a blank using the appropriate buffer. The blank for colorimetric assays is generally dH
2
O.
- Pedestal Option: Pipette 2 µL of dH
2
O onto the bottom pedestal, lower the arm and click Blank.
- Cuvette Option (Model 2000c only): Insert the cuvette noting the direction of the light path indicated by
the etched arrow. The optical beam (2 mm) is directed 8.5 mm above the bottom of the cuvette. Refer to
the cuvette manufacturer for volume recommendations.
Note: The arm must be down for all measurements, including those made with cuvettes. It is
recommended that cuvettes be removed from the instrument prior to making a pedestal
measurement to ensure that the pedestal arm can move to the proper starting position.
6. Under the Standards tab, highlight a standard and load as described for the blank above. Click Measure to
initiate the measurement. Be sure to measure all standards prior to measuring samples.
7. After all of the Standards have been measured, click on the Samples radio button. Enter a sample ID. Load
2 µL of sample when using the pedestal or insert the cuvette. Click Measure to initiate the measurement. It
is not necessary to blank the instrument between the standard and the unknown sample measurements.
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