GST Agarose

Paginas :

Procedure for Affinity Purification Using Gravity-flow Column (2 mL)
Note: Adjust volumes of sample and buffers accordingly.



Additional Materials Required:

• Binding/Wash Buffer: 0.1M sodium phosphate, 0.15M NaCl, pH 7.2

• Elution Buffer: 6M guanidine•HCl

• Storage Buffer: 0.05% sodium azide





A. Affinity Purification

Note: Perform chromatography at 4°C.

1. Resuspend product and dispense appropriate volume into Gravity-flow Column. Instructions below are for a 2 mL column.
2. Remove top cap and twist off bottom tab from column. Place column in a test tube and allow storage solution to drain.

3. Equilibrate column with 10mL of Binding/Wash Buffer. Do not allow resin to run dry; always recap column or proceed to the next step as soon as solution drains down to top of resin bed.

4. Mix equal volumes of antiserum and Binding/Wash Buffer. For optimal results, mix 3mL antiserum with 3mL Binding/Wash Buffer.

5. Apply buffered sample to the column.

6. Collect 0.5-1.0mL fractions. Monitor fractions by measuring the absorbance at 280nm relative to a buffer blank. All fractions above baseline contain protein without antibodies against GST.

7. Wash the column with 10mL of Binding/Wash Buffer, continuing to collect fractions.

8. Pool fractions that contain protein.



B. Elution of Bound Anti-GST Antibody

1. Apply 10mL of 6M guanidine•HCl to the column. Collect 0.5-1.0 fractions. These fractions will contain undesired antibodies that recognize the fusion protein′s GST portion.

2. Measure the absorbance of fractions at 280nm to verify the presence of the undesired anti-GST.



C. Column Regeneration and Storage

Note: To regenerate column, the anti-GST antibody must be eluted as described in Section B.

1. Apply 10mL of water, followed by 10mL of the Binding/Wash Buffer.

2. For storage, wash the column with 10mL of 0.05% sodium azide in water or buffer.

3. Cap column when approximately 1mL of solution remains above the top porous disc.

4. Store column upright at 4°C. Immobilized GST can be regenerated and reused a minimum of nine times.



Additional Information/Tips:

• Remove air bubbles from columns to restore flow rate.

• Degas buffers for use in affinity and gel filtration columns.

Aparência :

Suspension

Estado físico :

Liquid

envie :

Soluble in cold water, and hot water.

Manutencao :

Store at 4° C