Abstract
A method for the positive selection of specific antibodies for target proteins expressed as fusion proteins for the production of antiserum is presented. As proof of concept, the fusion protein FLAG::His::GFP::His::FLAG was expressed in Escherichia coli, purified, and used for the immunization of rabbits. The obtained serum was precleared via protein A affinity. A CusF::FLAG fusion protein was expressed in the periplasm of E. coli and purified. GFP without tags was also expressed in E. coli and purified via organic extraction. These proteins were then coupled to NHS-activated sepharose and used for the positive selection of Anti-GFP and Anti-FLAG antibodies. The obtained sera were tested for their specificity against different protein samples and fusion proteins in Western blots. A high specificity of the antibodies could be achieved by a single affinity chromatography step. In general, we advise to express the target protein with different tags and in different E. coli compartments for antibody production and affinity chromatography.
Highlights
Antibodies are important tools in many areas of molecular biology research but are increasingly used in medicine [1]
In order to increase the amount of antibodies against the FLAG-tag, we added a FLAG-tag at the N-terminus and at the
It was found that E. coli contains proteins with consecutive histidine residues, which will be co-purified by immobilized metal affinity chromatography [15], especially if the expression level of the fusion protein is low
Summary
Antibodies are important tools in many areas of molecular biology research but are increasingly used in medicine [1]. They can be produced by immunizing rabbits or other mammals or hens. One problem in obtaining antibodies is often the production of the antigen that is needed for immunization This can be the case if the antigen, usually a protein, is only expressed at very low levels. Purifying such proteins can be very time consuming, requiring months or even years of work. Even the target protein might be difficult to obtain
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