Antibodies can be effectively removed from a complex mixture containing other proteins through a process called affinity chromatography. This technique relies on the ability of antibodies to bind specifically to certain molecules, such as immobilized proteins like Protein A or Protein G.
A common approach involves using Protein A affinity chromatography. Here's how the antibodies are captured and then removed from the column in a purified state:
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Binding: The protein mixture is loaded onto a column where Protein A is attached to a solid support (the matrix). Antibodies in the mixture bind specifically to the immobilized Protein A. Other proteins that do not bind flow through the column and are washed away. This step effectively removes the antibodies from the rest of the sample by capturing them on the column.
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Washing: A washing buffer is passed through the column to remove any non-specifically bound proteins, ensuring high purity.
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Elution: To detach the captured antibodies from the Protein A and recover them, an elution buffer is used. According to the provided reference, this crucial step involves:
- Adding an acidic elution buffer (for example, 0.1 M glycine-HCl, pH 2.8).
- Collecting small fractions of the solution that comes off the column.
- The low-pH conditions cause the antibody to dissociate (release) from the immobilized Protein A.
- The purified antibody (IgG) is then recovered in these collected fractions.
This elution step effectively separates the antibody from the Protein A matrix, yielding a purified antibody preparation that has been successfully removed from the initial protein mixture.
Elution Step Details
| Action | Description | Purpose |
|---|---|---|
| Add Acidic Buffer | Apply buffer like 0.1 M glycine-HCl (pH 2.8) | Create conditions for antibody release |
| Dissociate Antibody | Low pH breaks the bond between antibody and Protein A | Release antibody from matrix |
| Collect Fractions | Gather small volumes of liquid leaving the column | Isolate the released antibodies |
| Recover Purified IgG | Obtain the antibody in a highly pure form | Get the final product after removal from mix |
By applying the elution step, the antibodies that were initially removed from the protein mixture by binding to the column are now recovered in a purified state, ready for subsequent use.
