Washing and Regenerating of Glutathione Agarose

 

Glutathione Agarose should be washed after each run, and preferably a given aliquot should only be used with the same protein. However, if the matrix becomes contaminated or shall be used with a different protein, a thorough cleaning procedure is recommended. This protocol delineates washing and regenerating procedures for PureCube Glutathione Agarose. Volumes are given in column bed volume (bv), i.e., 10 bv calls for 10 mL of buffer for a 1 mL column bed volume.

Wash Buffer, 100 mL

 
ComponentFinal concentrationMolecular weight (g/mol)Stock concentrationAmount needed for stockStock needed for buffer
Tris-HCl, pH 7.4 125 mM 121.14 0.5 M 30.29 g/500 mL 25 mL
NaCl 150 mM 58.44 5 M 146.1 g/500 mL 3 mL
DTT 1 mM 154.25 1 M 1.54 g/10 mL 100 µL
Instructions: Add water to a total volume of 100 mL.
 
   
 A.Wash protocol - Recommended after each use
 
 
Note: "bv" refers to column bed volume, i.e., 10 bv calls for 10 mL of buffer for a 1 mL column bed volume.
  1. After the elution step, add 10 bv Wash Buffer and allow the volume to completely flow through the matrix. The resin is now ready to be reused.
 B.Wash and store
 
 
  1. After the elution step, add 10 bv Wash Buffer and allow the volume to completely flow through the matrix.
  2. Rinse the column again with 10 bv water.
  3. Finally, add 10 bv 20% (v/v) ethanol and allow the majority of the volume to drip out of the column. Store at 2 - 8 °C.
 C.Intensive cleaning 
 
 
  1. After the elution step, add 10 bv Wash Buffer and allow the volume to completely flow through the matrix.
  2. Rinse the column again with 10 bv water.
  3. Wash with 2 bv of one of the following solutions. Do not combine the chemicals!
    1 M Sodium acetate, pH 4.0
    or 0.1 M Sodium hydroxide
    or 0.1 M Hydrochloric acid (HCl)
    or 70% (v/v) Ethanol
    or 1 % SDS
    or 1 % Triton X-100
  4. Rinse the column again with 10 bv water
  5. Wash the column with 10 bv Wash Buffer
  6. Repeat step 3-5 with a different chemical if necessary.
  7. The resin is now ready to be reused.
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