Protein A Resin

Protein Purification Protocol:

Buffer preparation
All water and buffers are recommended to be filtered with a 0.22 µm or 0.45 µm filter prior to use. Equilibration/Binding buffer: 20 mM Na2HPO4, 0.15M NaCl, pH 7.0-7.4. Elution buffer: 0.1M Glycine, pH 3.0. Neutralization Buffer: 1M Tris-HCl, pH 8.5. Storage buffer: 20% ethanol in PBS.

Column Packing
1. Assemble the column according to the column instructions. Fill the column with desired volume of slurry suspended in packing solution (20% ethanol in PBS). Let the flow run until the bed consolidates. Make sure no air is trapped in the bed.
2. Protein A columns can be installed on medium or low-pressure chromatography systems, ensuring that the pressure is less than 0.3MPa.
3. Equilibrate the column with 5-10 column volumes (CV) of equilibration buffer.

Sample preparation
Make sure that the sample solution has the appropriate ionic strength and pH value before loading the column. Serum samples, ascites fluid or cell culture media can be diluted with equilibration buffer, or the sample can be dialyzed with equilibration buffer. It is recommended that samples be filtered with a 0.22 µm or 0.45 µm filter or centrifuged to remove micro particles prior to loading. It is recommended to dilute the sample with the target IgG concentration around 2mg/ml.

IgG Purification Procedure
1. Load the samples at a flow rate of 0.2ml/min(for 1ml column, with residence time about 5 minutes). The maximum volume of the sample to be loaded is calculated according to the concentration of the target IgG and the DBC (dynamic binding capacity) of the column: total target IgG does not exceed the 80% of the DBC.
2. Wash the column with 5-10 CV Equilibration buffer until OD280 is at the baseline.
3. Elute the column with 5-10 CV Elution buffer until OD280 is at the baseline. Collect the fractions in tubes.
4. Pool the fractions with target IgG and quantification. Add neutralization buffer to PH 7.0-7.5.

Column Wash
1. Wash the column with 5-10 CV equilibration buffer at flow rate of 0.2ml/min (for 1ml column).
2. Wash the column with 5-10 CV storage buffer (1XPBS with 20% ethanol), then store at 2-8°C.

Column Regeneration
1. Regenerate the column with 3-5 CV elution buffer.
2. Equilibrate the column with 3-5 CV equilibration buffer.

Clean in place (CIP)
Cleaning-in-place can effectively remove accumulated contaminants on the resin to recover the binding capacity. Perform clean-in-place as the following procedure:
1. Wash with 0.2 M NaOH for 2 CV.
2. Wash with 6 M guanidine hydrochloride for 2 CV.
3. Wash with 0.1 M phosphoric acid for 2 CV.
4. Equilibrate with equilibration buffer for 2 CV.