Affinity chromatography, also known as affinity purification, uses the specific ways molecules stick to each other. A particular ligand is chemically attached to a solid support, or “coupled,” so when a complex mixture is passed over the column, only the molecules with a strong affinity for the ligand will stick to it. After other parts of the sample are washed away, the bound molecule is removed from the support, separating it from the rest of the sample.
What is the Affinity Purification Antibody Protocol?
Affinity purification antibody protocol techniques are used in several ways to clean up antibodies. Most of the time, small amounts of serum, ascites fluid, or culture supernatant are used to make antibodies in the lab. These must be partially or fully purified depending on how the antibody will be used for different tests and detection methods.
Understanding Antibody Purification
Affinity purification antibody protocol involves the selective enrichment or specific isolation of antibodies from serum, ascites fluid, or the cell culture supernatant of a hybridoma cell line. Methods for purification range from very basic to very specific, and they can be put into the following groups:
• Differential precipitation, size-exclusion, or solid-phase binding of immunoglobulins based on size, charge, or other chemical properties that most antibodies in a sample share. This separates a group of proteins from the sample, which includes the immunoglobulins.
• Class-specific affinity binds antibody classes (like IgG) to biological ligands (proteins, lectins, etc.) that are immobilized and have a specific relationship with immunoglobulins. This separates all antibodies of the target class, no matter how specific they are for an antigen.
• Antigen-specific affinity is the process of only removing antibodies from a sample that bind to a specific antigen molecule through their antigen-binding domains. This separates all antibodies that attach to the antigen, no matter their class or isotype.