Bio-Rad Profinia™ Protein Purification Instrument - Manual (Page 67)

Note: The peak detect delay and max peak volume parameters can only be edited in the
program methods mode (see section 7.3.1).
• Peak detection sensitivity — settings range between 0.1 (most sensitive) and 10
(least sensitive); the default setting is 1. Peak detection sensitivity is a global setting
accessible in the Data/Utilities menu for both Bio-Rad methods and program methods
modes. The peak detection sensitivity parameter programs the Profinia instrument to
adjust the automatic peak detection algorithm to various peak shapes and sizes in the
event the default setting does not recognize a peak during the specified time window. In
general, use lower numbers (more sensitive) if a small protein peak is expected.
Note: Using more sensitive settings for peak detection may lead to activation of automatic
peak detection from small UV deviations not associated with the protein peak.
Manual Peak Detection
In addition to automatic peak detection, the instrument provides the option to manually
detect and divert the protein peak during affinity and desalting elution steps. Manual peak
detection can be used for:
• Purifications where the target protein is in a very low concentration (<1 mg/ml) or has a
very low extinction coefficient. Cold room purification and using program methods with
the protein elution at a flow rate lower than 0.5 ml/min may generate UV trace slopes
not steep enough to be detected by the Profinia peak detection algorithm. If these types
of sample and running conditions are typical, then it is recommended to adjust the peak
detection setting to the highest sensitivity: 0.1. However, the Bio-Rad methods default
value for the max peak volume is intended as a safety for cases where the peak is not
automatically detected, for example, for very low expression proteins. When this value
is reached the method will advance to the next step to ensure that the region where the
protein is eluting will be collected
• Overriding the automatic peak detection to collect the highest possible protein
concentration by manually selecting the peak section with the highest absorbance values.
To override the automatic peak detection, set the peak detect delay time and max peak
volume to values well beyond the point the peak is expected to elute. Editing these
values is possible in program methods mode only (see section 7.3.1 for more details).
The manual peak detect and manual peak divert buttons will display on the screen during
the appropriate protein elution steps only.
Using manual peak detect:
When the protein peak starts to elute, touch the Manual Peak Detect button to
immediately advance to the next step without waiting for automatic peak detection or the
max peak volume setting.
Using manual peak divert:
You have the option to minimize the volume in which the purified protein is collected.
Diverting fraction collection before the peak trailing ends decreases the total yield and
increases protein concentration.
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