Bio-Rad Profinia™ Protein Purification Instrument - Manual (Page 188)

Topic Tips and Recommendations
Sample
Application
Load and Flow Rate The yield of the target protein is dependent on the flow rate at
Dependency which the sample is applied to the column. Lowering the flow rate
may significantly improve yield.
For viscous samples, choose the low flow sample application
parameter in order to avoid exceeding the pressure limits of the
Profinia system. It may be necessary to improve the efficiency of
cell lysis of highly viscous samples by:
•
Increasing sonication time
• Clarifying and filtering the sample prior to use in the Profinia
system
• Adding nucleases or trying a different lysis method
Slowing down the flow rate of the sample loading step allows the
target protein more time to diffuse within the column and find a
binding site. This is especially true when purifying protein by
denaturing IMAC. Denaturing IMAC buffers are very viscous, so
slowing down the flow rate for the sample loading and wash-1
steps can greatly increase yield.
Load Volumes For optimal recovery on the Profinia system, use load volumes
5 ml or greater. Ideally, dilute small/concentrated samples from
<5 ml to >10 ml to maximize yield.
Large Sample When low protein expression requires large culture volumes
Volumes (>2 L of starting culture), bacterial pellets can be lysed at 5:1 (v/w)
ratio to help keep the total sample volume equal to the maximum
load volume allowable in the preset Bio-Rad Methods (50 ml).
Alternatively, a custom Program Method can be used along with
an external sample container (see Section 11.2.2 on pg. 128) to
load volumes up to a maximum of 999 liters.
Proper Load Ensure the sample is free from particulate matter by filtering it
Volume Not Applied through a 0.22 or 0.45 µm filter immediately prior to
chromatography.
Check/change the inline filter. Change the inline filter on a regular
basis if running large sample volumes.
Periodically run the short-term system wash with 20% ethanol to
ensure optimal system performance and flow rates.
Sample
Analysis
Low Target Protein The yield of the target protein is dependent on the flow rate at
Yield which the sample is applied to the column. Lowering the flow rate
may significantly improve yield.
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