General Instructions for Affinity Purification Using HiTrap® Columns

Appendix 4, Extracted from Affinity Chromatography Vol. 1: Antibodies, GE Healthcare, 2016
 

Alternative 1. Manual purification with a syringe

Using HiTrap columns with a syringe.

Fig A4.1. Using HiTrap® columns with a syringe. (A) Prepare buffers and sample. Remove the column’s top cap and twist off the end. (B) Equilibrate the column, load the sample and begin collecting fractions. (C) Wash and elute, continuing to collect fractions.
 

  1. Fill the syringe with binding buffer. Remove the stopper and connect the column to the syringe (use the connector supplied) “drop to drop” to avoid introducing air into the column.
  2. Remove the snap-off end at the column outlet.
  3. Equilibrate the column with 5 column volumes of binding buffer.
  4. Apply the pretreated sample using a syringe fitted to the Luer connector on the column. For optimal results, use a flow rate of 0.2 to 1 ml/min (1 ml column) and 0.5 to 5 ml/min (5 ml column) during sample application*.
  5. Wash with 5 to 10 column volumes of binding buffer or until no material appears in the effluent. Maintain a flow rate of 1 to 2 ml/min (1 ml column) and 5 to 10 ml/min (5 ml column) for washing. Optional: collect the flowthrough (in 1 ml fractions for the 1 ml column and 2 ml fractions for the 5 ml column) and reserve until the procedure has been successfully completed. Retain a sample for analysis by SDS-PAGE to measure the efficiency of protein binding to the medium.
  6. Elute with 5 to 10 column volumes of elution buffer. Maintain a flow rate of 0.2 to 1 ml/min (ml column) and 0.5 to 5 ml/min (5 ml column) for elution.
  7. After elution, regenerate the column by washing it with 3 to 5 column volumes of binding buffer. The column is now ready for a new purification.

* 1 ml/min corresponds to approximately 30 drops/min when using a syringe with a HiTrap® 1 ml column; 5 ml/min corresponds to approximately 120 drops/min when using a HiTrap® 5 ml column.

 

For large sample volumes, a simple peristaltic pump can be used to apply sample and buffers.

Alternative 2. Simple purification with ÄKTAprime plus

ÄKTAprime plus contains preprogrammed templates for purification of IgG, IgM, and IgY using  the appropriate HiTrap® columns.

Prepare at least 500 ml of each buffer.

  1. Follow instructions supplied on the ÄKTAprime plus cue card to connect the column and load the system with binding buffer.
  2. Select the Application Template.
  3. Start the method.
  4. Enter the sample volume and press OK to start.

Typical procedures using AKTAprime plus.

Fig A4.2. Typical procedures using ÄKTAprime plus. (A) Prepare the buffers. (B) Connect the column. (C) Prepare the fraction collector. (D) Load the sample.

 

Materials

     
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