His tag is a short amino acid sequence consists of histidine (His) residues in recombinant proteins. Immobilized metal-ion affinity chromatography (IMAC) and monoclonal antibody affinity chromatography are used for the purification of His-tagged proteins from eukaryotic and prokaryotic expression systems. After purification, the His-tagged proteins can be specifically detected by anti His tag monoclonal antibody (mAb).
GenScript His Tag Protein Detection Kit is developed for the detection of His-tagged protein expression and semi-quantitation using a standard of His-tagged protein of known molecular weight (M.W.). This kit contains all reagents and buffers required for His tag detection, suitable for most systems, such as bacterial, yeast, mammalian cell lysates and cell culture supernatants.
This kit is based on competitive ELISA, where a His-tagged protein (M.W. 12.7kD) is pre-coated on the microwell plate.Series dilutions of His-protein standards (11.3kDa) and samples are added to each test well, then, anti-His tag mAb is added to the plate. His-tagged target protein in solution and pre-coated His-tagged protein compete to bind the antibody. After washing steps, HRP-conjugated secondary antibody is added to each well to react with anti-His tag mAb. The final step involves the addition of ™B substrate, followed by the addition of stop solution for signal development. The OD reading will indicate expression level of His-tagged proteins. In certain cases His-tagged protein in the sample may be semi-quantified by comparing to the standard curve generated from known His-tagged protein. This kit also offers a His-tagged protein stock for customer recovery verification assay.