Bio-Rad Profinia Protein Purification System Kits - Manual (Page 11)

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Select Sample Parameters Screen
The Select Sample Parameters screen
gives users the option of increasing/
decreasing the rate of flow or the
number of column volumes of wash
used. The selections in Figure 4 are.
• Flow rate: Standard*
• Wash time: Standard
* Standard flow rates for IMAC purifications are
2 ml/min. Proteins that are very large (>100 kD),
have His residues that are not fully exposed, or require
cold room purifications, typically bind with slower
kinetics. In these situations, the slow flow rate
option (0.5 ml/min) increases binding efficiency.
Enter Run and Sample Information
Screen
Users can customize the method by
adding user-specific details that
distinguish samples from one another
(Figure 5). Such details may include
user and run name, sample name
and volume, and A
280
of a 1 mg/ml
protein solution. Selections in Figure 5
are:
• Username: Anonymous
• Run name: UNTITLED
• Lot tracking: BLANK
• Sample 1 name: 3/4 vial51kd
• Volume: 5.00 ml
• A
280
of 1 mg/ml*: 1.33
*All proteins absorb UV light based upon amino acid
composition. The A
280
of a 1 mg/ml protein solution
can be calculated if the specific amino acid sequence
is known. For 51-D in the control lysate, the OD
280
of a
1 mg/ml solution is 1.33. By entering this value,
the Profinia instrument automatically calculates and
converts the concentration of the purified protein from
the absorbance values. The concentrations given by
the Profinia system are estimates of the purified protein.
Analytical determination of protein concentration using
a spectrophotometer or Bradford analysis following
purification is always good practice.
Fig. 5. Run and sample information
selection screen.
Fig. 4. Method selection for sample flow
rate and wash times.
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