Bio-rad Profinity IMAC Resins User Manual download pdf (Page 38)

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

)

– 300 mM NaCl

– 5 mM imidazole

– Up to 8 M urea

Adjust to pH 8.0.

• Wash buffer

– 50 mM sodium phosphate (NaH

)

– 300 mM NaCl

– 10 mM imidazole

– Up to 8 M urea

Adjust to pH 8.0.

• Elution buffer

– 50 mM sodium phosphate (NaH

)

– 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

10001677B.qxd 1/28/2005 12:42 PM Page 37

1 2 ... 33 34 35 36 37 38 39 40 41 42 43 ... 51 52

Comments to this Manuals

No comments

Bio-rad Profinity IMAC Resins User Manual Page 38

Comments to this Manuals

Related products and manuals for Accessories for water Bio-rad Profinity IMAC Resins