This article describes a method for purifying monoclonal antibodies using affinity chromatography with Protein A. The process involves loading a culture supernatant onto a column, washing away contaminants, and eluting the antibodies for further analysis.
Take an affinity chromatography column containing agarose beads conjugated to Protein A, an antibody-binding protein.
Add a binding buffer with a neutral pH to equilibrate the column.
Load the culture supernatant from an antibody-producing cell culture, containing monoclonal antibodies and contaminating cellular proteins.
The neutral pH facilitates protein A binding to the Fc region of the antibodies while the contaminants flow through.
Measure the UV absorbance of the flow through to create a chromatogram. A high absorbance during sample loading indicates unbound proteins in the flow through.
Wash the column with the binding buffer to remove non-specifically bound contaminants.
Add an elution buffer with a low pH to disrupt the Protein A-antibody interaction. Collect the eluted antibodies by monitoring the peak in absorbance.
Neutralize the pH to prevent antibody degradation and transfer into a centrifugal filter.
Centrifuge to concentrate the antibodies and resuspend in a buffer for downstream assays.
After preparing buffers and initializing the FPLC system according to the text protocol, use the method editor to set up the run steps as follows. Equilibrate the system with five column volumes or CV of binding buffer.
Load the sample onto the column and collect the sample flow through, and then use five CV of binding buffer to wash the system. Use five CV of elution buffer to elute the column via isocratic fractionation. and collect the purified antibody samples as fractions using the fraction collector.
Finally, equilibrate the system with five CV of binding buffer and collect the flow through into a waste container. Once the method setup is complete, specify the volume of conditioned medium to be applied to the column and save the method.
Next, submerge the sample pump tubing into the vessel containing the conditioned medium. Then, prepare a separate container to collect sample flow through via the specified outlet tubing. Insert collection tubes in the fraction collector and add neutralization buffer to each collection tube. In the system control module of the software, open the method to be used. Then click Start to initiate the run.
When the purification is complete and the evaluation module, check the resulting chromatogram. Combine all the protein-containing fractions into a tube, and then use PBS and a centrifugal filter device with a 30-kilodalton cutoff to concentrate the sample and carry out buffer exchange. Finally, use the BCA assay to measure the antibody concentration according to the manufacturer's instructions.
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