Bio-Rad Profinia™ Protein Purification Instrument - Manual (Page 102)

5. Touch the Start button; the system pumps water from the water bottle to the waste
outlet to remove air from the system and the delivery tube. When the step is finished,
the screen displays “Priming Complete” and additional instructions.
6. Remove the waste line from the waste bottle and place the waste line in an empty
graduated cylinder.
7. Touch Start to pump deionized water through the system at the flow rate and for the
duration set in step 4.
8. When the test is complete the system displays "Flow Rate Check Completed". Note the
expected quantity on the screen. The actual quantity delivered when bypassing the
cartridged during the flow rate check can be up to 7% over the expected value if the
system is working optimally. The additional restriction from directing the flow through
the cartridges during the method will result in the expected quantity displayed on the
screen.
If the quantity is less than the displayed expected quantity, change the inline filter disk and
make sure that all the air has been purged from the system before repeating the test
procedure.
If you continue to have problems with system flow rates, clean or replace the pump check
valves (Section 13.2.1) or replace the pump seals (Section 13.2.2). If the system still does
not operate normally, contact your local Bio-Rad office for assistance.
9.2.3 Calibrating UV Monitors
You may wish to calibrate the UV monitors using a protein similar to your protein of interest
for improved quantitation. The following materials are required to calibrate the UV monitors:
Protein solution of known absorbance — prepare a protein solution and measure its
absorbance in a spectrophotometer at 280 nm. The absorbance value for a 1 cm pathlength
will be entered prior to the calibration process. Note:
• Choose a solution that has an absorbance reading less than 2 AU at 280 nm, for a
1 cm pathlength on the spectrophotometer
• If you use a cuvette that is less than 1 cm pathlength, correct the absorbance
reading to 1 cm. For example, if you use a 2 mm cuvette, multiply the reading by
5 and enter this value in the calibration screen
• Choose a solution that has an absorbance reading less than 1 unit on the UV monitor
(the instrument has a 2 mm pathlength)
• A protein concentration of 1–2 mg/ml is a suggested starting point
Deionized water — 45 ml in a 50 ml sample tube; 500 ml in the diluent (water) bottle for
placement in the system’s Di water port
20% ethanol solution — 100 ml in a Profinia 125 ml buffer bottle
To calibrate the UV monitors:
1. Fill a 125 ml Profinia buffer bottle with 20% ethanol and place it in buffer position 8.
2. Prepare the standard protein solution and measure its absorbance at 280 nm. Correct
the absorbance value to 1 cm pathlength.
3. Fill a 50 ml sample tube with 45 ml of this UV absorbance standard solution, and place
it in sample position 1.
95