IAC - Immunoaffinity chromatography

Overview

Immunoaffinity chromatography is a technique used to purify a specific protein or molecule from a complex mixture using an antibody that selectively binds to the target. The antibody is immobilized on a solid support material, such as beads or a column, and the sample is passed through the column. The target protein or molecule binds to the antibody and is then eluted from the column for further analysis. It is a highly specific and sensitive method for purifying proteins and other molecules from biological samples.

Required materials

• Column packed with protein A/G resin

• PBS buffer

• Blocking buffer

• Primary antibody

• Sample containing antigen

• Secondary antibody conjugated to HRP

• Elution buffer

• Collection tubes

Protocol

1. Equilibrate the protein A/G resin in PBS buffer to remove any contaminants.

2. Rinse the column with a blocking buffer to block any nonspecific binding sites.

3. Dilute the primary antibody in the blocking buffer and load it onto the column.

4. Allow the antibody to bind to the protein A/G resin first for at least 30 minutes.

5. Dilute the sample in the blocking buffer and load it onto the column.

6. Allow the sample to flow through the column and interact with the immobilized primary antibody.

7. Wash the column with PBS buffer to remove any unbound material and contaminants.

8. Elute the antigen: Elute the antigen of interest by using an elution buffer to disrupt the antigen-antibody complex.

9. Collect the eluted fraction in a collection tube.

10. Dilute the fraction in the detection buffer and apply it to a detection system, HRP conjugated secondary antibody. This detection system will generate a signal that can be measured, indicating the presence of the antigen.

11. Repeat steps 7-11 if necessary to further purify the antibody.

12. Store the eluted antigen in the appropriate buffer and conditions until further use.

Results

A few types of results can be gathered using immunoaffinity chromatography:

• Purification of specific protein or molecule, Immunoaffinity chromatography can be used to purify a specific protein or molecules from a complex mixture by selectively binding to the protein or molecule of interest: the technique can remove contaminants and unwanted molecules, resulting in a highly purified sample.

• Quantification of a molecule or protein: Once a protein or molecule is captured by the immunoaffinity column, it can be quantified using various methods, such as ELISA, this allows for accurate measurement of the amount of protein or molecule present in a sample.

• Identification of interacting proteins and molecules: By capturing proteins or molecules of interest using immunoaffinity chromatography. This can give insight into the function and mechanisms of the protein or molecule being studied.