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Enzyme Purification by Immunoprecipitation



Creative Enzymes supports researcher from various industries with high-quality products and services. Based on decade-long development of our unique techniques, we are offering reliable enzyme purification services with a wide range of choices of available methods. Immunoprecipitation (IP) is one of the most efficient methods for the isolation of enzymes and enzyme complexes. Our services include the design, standard operations, and the quality inspection.

Immunoprecipitation is a method for isolating small amounts of target protein from complex samples such as cell lysates, serum, and tissue homogenates. The antibodies used in IPmay be polyclonal or monoclonal and may recognize the enzyme of interest, a particular post-translational modification, or an epitope tag if the protein is overexpressed. There are three widely used methods of immunoprecipitation.

In traditional method, antibodies are first incubated with the sample containing the target enzyme (antigen). After the antigen-antibody complex is formed, it is bound to Protein A or Protein G beads, which are typically cross-linked agarose. Subsequently, the beads and the sample are centrifuged to pellet the captured immune complex. However, the problems of this purification method include the contamination of the target protein with the IP antibody, the destruction of costly IP antibody and sample loss incurred when pipetting or removing supernatants from small resin pellets. Besides, contamination is most problematic if the antigen or its interacting partner has similar molecular weights with the antibody heavy and light chains since these bands will co-migrate in SDS-PAGE.

This method allows the crosslink of the antibody to Protein A or G beads, resulting in a permanent affinity support with the antibody properly oriented to bind the target antigen. This method is a preferable use when the amount of antibody is in short supply or expensive because the antibody: Protein A or G resin can be reused.

In this method, the antibody is coupled directly to the agarose beads, eliminating the need for a linker or Protein A or G. This method is especially beneficial when using antibodies that do not bind strongly to Protein A or G. However, this direct affinity strategy results in a random orientation of the antibody on the beads. This probably causes a reduction in antibody binding efficiency under certain conditions. The advantage of direct affinity method is that the eluted fraction is not contaminated with antibody heavy and light chains, and the resin may be reused several times.

Enzyme Purification by Immunoprecipitation Figure 1: The strategies for preparing immunoprecipitation matrixes.

Figure: Comparison of antigen binding efficiency of  the three IP methods. Figure: Comparison of antigen binding efficiency of the three IP methods.

A variety of factors may influence the purification process, including IP antibody class, the properties of the purified protein as well as the available and cost of the antibody. Creative Enzymes takes fully consideration of these factors. We evaluate the purification strategy before perform the purifying process to make sure the most suitable protocol will be used for each enzymes. Our technical team is proficient in establishment of proper methods and accomplishment of reliable operations ensure the high quality of the purification services. This custom service will accelerate research and commercialization in any type of enzyme applications.



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