Bio-Rad Profinity IMAC Resins - Manual (Page 38)

34
Section 13
Sample Preparation-Size Spin-Column
Purification of His-Tagged Proteins Using
Denaturing Conditions
Materials
Reagent
• Binding buffer (urea-based)
–
50 mM sodium phosphate (NaH
2
PO
4
)
– 300 mM NaCl
– 5 mM imidazole
– Up to 8 M urea
Adjust to pH 8.0.
• Wash buffer
–
50 mM sodium phosphate (NaH
2
PO
4
)
– 300 mM NaCl
– 10 mM imidazole
– Up to 8 M urea
Adjust to pH 8.0.
• Elution buffer
–
50 mM sodium phosphate (NaH
2
PO
4
)
– 300 mM NaCl (pH 6.8 or higher)
– 250 mM imidazole
– Up to 8 M urea
Adjust to pH 8.0.
• Optimized wash buffer (optional, see Section 10)
A wash buffer containing slightly less imidazole than necessary to elute
the target protein may be used to increase the stringency of the wash
step. Refer to Section 10, Medium-Pressure Column Purification — Using
an Imidazole Gradient to Determine Optimal Purification of His-Tagged
Proteins.
Once the concentration of imidazole in the wash step is determined using
medium-pressure column chromatography, a stepwise elution step may
be carried out as indicated in this protocol.
Equipment
• Sample preparation sized IMAC spin column (as prepared in Section 5)
(for example, Micro Bio-Spin column
™ , catalog #732-6204)
• Plasticware, 2 ml capped, and 2 ml capless tubes
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