Millipore DDHW00010-WW, Руководство пользователя

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Direct Detect® Spectrometer User Guide 43
14.5 Protein Measurement Verification
,
continued
4. Pipette exactly 2 µL of sample to sample positions 2 through 4. Card positions can be
labeled if desired.
5. In the Direct Detect® Software main screen, complete the measurement setup as
indicated in Figure 44. Choose
NIST BSA AM1
from the protein drop down menu and
check the
Dry sample card
box. Sample location 1 should be blue, indicating that the
buffer solution is in this location. Sample locations 2–4 should be green, indicating
that there is a sample to be measured.
Figure 44. Sample information
6. Follow the instructions in section 10.2 for inserting the sample card into the
instrument, then click on the button.
7. The sample measurement results are displayed on the
Last Sample Result
tab of the
main screen.
8. Measure the BSA standard concentration using a UV reader at 280 nm.
DO NOT use standard curves built into the UV reader. Use the sequence-based
extinction coefficient 42,925 M
-1
cm
-1
or ABS 1 mg/mL = 0.646. Zero the UV instrument
with the correct buffer and read absorbance at 280 nm.
9. Manually calculate the correct concentration using the sequence-based numbers. The
system passes the protein verification test if the results from the UV reader and the
Direct Detect® system are within 10% of one another. Document results on the
IQ/OQ/PQ Report Summary.
NOTE:
The Direct Detect® system comes pre-loaded with standard curves that
were generated from phosphate-buffered saline (PBS). Analysis from buffers
like NaCl with NaN
3
delivers accurate quantification if the measurement is
performed in a room with humidity level below 35%. Humidity levels above
35% will affect sample drying, resulting in artificially increased concentration
estimates.