Bio-Rad Profinity IMAC Resins - Manual (Page 41)

37
Section 14
Batch-Mode Purification of Histidine
Proteins — Gravity Flow
The following guideline is a general procedure for batch-mode binding, which is
an effective means for purification when the recombinant His-tagged protein is
either expressed at low levels or when the tag itself may be inaccessible.
Recommended Columns
The following Bio-Rad columns may be used for gravity-flow chromatography. They
are empty and can be filled with a support of choice.
• Poly-Prep ® columns, for bed volumes up to 2 ml of chromatography
support and 10 ml sample
• Econo-Pac ® columns, for bed volumes up to 20 ml
• Glass Econo-Column ® columns, 5–170 cm long and 0.5–5.0 cm in
diameter
Materials
Reagents
• Binding/wash buffer
–
50 mM sodium phosphate (NaH
2
PO
4
)
– 300 mM NaCl
– 5 mM imidazole
Adjust to pH 8.0.
• Elution buffer
–
50 mM sodium phosphate (NaH
2
PO
4
)
– 300 mM NaCl
– 500 mM imidazole
Adjust to pH 8.0.
Equipment
• IMAC resin
• Sample preparation-sized IMAC spin column (as prepared in Section 5)
(for example, Micro Bio-Spin columns, catalog #732-6204)
• Plasticware, 2 ml capped, 2 ml capless tubes
Biological Sample
• Clarified lysate (as prepared in Section 7)
The binding capacity of the Profinity ™ IMAC resin is
≥15 mg His-tagged
protein per ml resin. Larger amounts of protein will require a larger column
to be used.
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