Bio-Rad Profinia™ Protein Purification Instrument - Manual (Page 189)

Topic Tips and Recommendations
Precipitation of Precipitation of the elution peak can occur when the eluted protein
Elution Peak is too highly concentrated. Load a lesser volume of sample or add
protein stabilization reagents such as glycerol (up to 10%),
reducing agents, and detergents to help prevent precipitation of
the eluted protein.
Target Protein in Check the pH of the sample after lysis and ensure that it is 7.0.
Flow Through His-tagged proteins will not bind under acidic conditions.
Keep the concentration of chelating agents, such as EDTA, to
1 mM. EDTA will strip the nickel-charged resin if used at too high
a concentration.
Low Purity of His- Increase the concentration of imidazole in the sample and wash
Tagged Target buffers to minimize binding by host proteins. Higher imidazole in
Protein the sample may decrease binding of His-tagged proteins, resulting
in lower yield. Optimization of imidazole concentration is necessary
to balance higher purity and yield.
Purification and
Buffer Kits
Proper Storage of All of the kits can be stored at 22°C (room temperature) for short
Solutions and Kits periods of time, either upon receipt or during normal usage. For
kits that have labile reagents (glutathione in GST kits), 4°C is the
recommended long-term storage temperature.
Once opened and used with the instrument, all reagents should
be stored at 4°C for up to 3 months. After insertion into the
instrument, the solutions are no longer sterile and require storage
at 4°C.
Once opened and used with the instrument, check the solutions
for particulates and clarity before reusing. If there is any indication
of particulates, contamination, or microbial growth, the solution
should be discarded.
Addition of The solutions are provided as concentrates, and are diluted by the
Additives or Profinia instrument. If another component need to be added to one
Reagents to the of the solutions (for example, a protease inhibitor), it should be
Solutions added as a concentrate and brought to the final concentration
of the solution (for example, final 2x concentration for the affinity
buffers). The volume to be added should be minimized so that it
does not dramatically alter the final concentration of the solution
labeled on the bottle.
Addition of Urea Urea and glutathione are supplied in convenient, premeasured
and Glutathione aliquots for each buffer bottle. These reagents should be added to
the buffers on the day purification starts. Any unused solution can
be stored at 4°C for up to 7 days.
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