Encyclopedia of Experiments: Immunology
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Transcript
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.
