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Isolation of Antibodies Against Different Protein Conformations by Immunoaffinity Chromatography (CAT#: STEM-ACM-0104-CJ)

Introduction

Antibodies that recognize conformational epitopes of natural proteins or linear epitopes of denatured proteins can be isolated from antisera containing a set of polyclonal antibodies that target different epitopes of an antigen using immunoaffinity chromatography. The method does not require immunization with an antigen of a certain conformation that is stabilized in some way (cross-linked, modified, or denatured).




Principle

Immunoaffinity chromatography is a method for separating target antibodies or antigens from a heterogeneous solution. It is column-based, which means that the solution is flowed through a column and eluted at the other end. The column is pre-functionalized with the capture antibody or antigen. The target protein is adsorbed onto the resin-bound capture protein and is retained in the column while the remaining solution is eluted. The fraction containing the target protein is later eluted and purified.

Applications

Biochemistry

Procedure

1. Preparation of Column: The column is loaded with solid support such as sepharose, agarose, cellulose etc.. Ligand is selected according to the desired isolate. Spacer arm is attached between the ligand and solid support.
2. Loading of Sample: Solution containing a mixture of substances is poured into the elution column and allowed to run at a controlled rate.
3. Elution of Ligand-Molecule Complex: Target substance is recovered by changing conditions to favor elution of the bound molecules.

Materials

• Sample: Plants; Natural Food; Protein; Drug; Pollutants; Blood; Saliva; Serum; Plasma; Antibodies; Viruses & More
• Equipment: Agarose; Silica gel; Aluminium oxide; Acrylate; Organic polymers; Wash Buffer
• (Optional): Ligand; Spacer arm; Column

Notes

1. Immunoaffinity chromatography is one of the most widely used affinity-derived procedures, and it permits the production of ligands when the needed ligand is unavailable.
2. In this method, the stationary phase consists of an antibody or an agent linked to antibodies. Due to the high specificity of antibodies, it is possible to isolate varied substances using this method.
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