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Posted on Thu, Apr 26, 2018

Elution Buffer

An elution buffer plays an essential role in every immunoprecipitation protocol or assay that requires the release of a target antigen from a capture antibody. Elution buffers are necessary in protocols utilizing a stationary affinity column, and are also required in protocols using mobile solid supports in solution.

The elution buffer interferes with and disrupts antibody-antigen interactions. The premise of an immunoprecipitation assay is that capture antibodies are immobilized on a support platform and specifically bind target antigens. These antibody-antigen complexes are then recovered either by centrifugation or magnetic separation, but the antigens need to be separated from the antibodies for examination by SDS-page. If the antibodies remain bound to the antigens then they will contaminate the SDS-page and complicate analysis by Western blot. 

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Elution buffers target antibody-antigen interactions

Elution buffers are chosen for their ability to interfere with ionic, hydrophobic, or hydrogen bonding. Some elution buffers are specially formulated for use in metal affinity chromatography where metallic ion interactions need to be disrupted. Additional consideration should be given to denaturation in applications where higher order protein structure needs to be preserved, such as co-immunoprecipitation. Non-denaturing elution buffers are ideal for those experiments.

The most common method of interfering with ionic interactions is to use an ionic elution buffer with a high salt content. NaCl solutions are often used. The pH of the buffer can also play a role as protein interactions are most efficient at physiological pH. Raising or lower the pH can help disrupt those interactions. One of the gentlest and most commonly used components in non-denaturing elution buffers is glycine-HCL with a very acidic pH of 2.5-3.0. This buffer disrupts the antibody-antigen interactions without denaturing the proteins. After the antigens are collected the pH is neutralized to prevent long-term damage to the proteins.

When hydrogen bonding is the dominant antibody-antigen interaction a chaotrope, or salt that interferes with hydrogen bonds, is used. Some examples of commonly used chaotropes are guanidinium choride and phenol.

When denaturation of the antigen or target proteins is not a concern or problem, then denaturing elution buffers can be used to improve capture efficiency and decrease antibody contamination. SDS is very common. Sodium deoxycholate is also used.

Elution buffer

Elution buffers for metal affinity chromatography typically contain a high concentration of imidazole to disrupt metallic binding sites. This strategy is common for recovery of histidine tagged proteins. During capture, the histidine tag binds to the metal ions immobilized on a support column. For recovery of the target protein, the imidizole has a higher affinity for the metal ions and kicks out the histidine tagged proteins. The proteins then move into solution and are eluted from the column where they can be collected for further analysis.  

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Purification IgG antibodies by protein A or G affinity is another good example of a situation where a denaturing elution buffer can be harmful to the final product. The conservation of an antibody’s tertiary structure is extremely important for the maintenance of its antigen recognition site. Antibody-antigen affinity is not a covalent bond; it is dependent on hydrophobic, hydrophilic, and ionic interactions. Therefore, an extremely acidic elution buffer will interfere with the chemical signature of the IgG’s antigen recognition site. Recombinant proteins have been engineered to address this problem. It has been found that the addition of histidine tags to the IgG recognition region of Protein G can increase the pH of dissociation. In this case, the surface of the histidine becomes positively charged as pH drops and the solution becomes more acidic. This postitive charge interferes with the IgG-protein G association and they more readily dissociate at a less acidic pH. This can help to preserve IgG antibody function after the purification process is complete.

One thing to keep in mind is that all elution buffers result in some loss of antibody or antigen function. Therefore, the ability to reuse antibodies and support platforms is limited. However, the commercial availability of immunoprecipitation capture antibodies, platforms, buffers, and kits makes this of minor concern.

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