![]() ![]() Especially in case of non-automated, manual fraction collection in chromatography, the workload to identify the His-tag containing fractions via SDS-PAGE and Western blot is significant. 1) to support the characterization of recombinant protein expression and subsequent purification steps. Here, we established a technique for an immediate detection of His 6-labeled proteins in crude biological samples (e.g., cell lysate for protein expression specimens) based on a 90 s immunoassay principle 8, 9 and an in-house developed monoclonal anti-His-tag-antibody.Ī fast homogeneous immunoassay based on Förster resonance energy transfer was developed for the detection of His-tagged proteins ( Fig. A fast mix-and-measure assay for specific detection of His-tagged proteins could therefore simplify the identification process of His-tag containing fractions dramatically. However, the characterization of all protein fractions via SDS-PAGE and, if needed Western blot, although being used as a standard procedure, is a time-consuming procedure, especially if it is only to identify the fractions containing the target protein. In some cases, additional Western Blot experiments may be performed, for which a variety of anti-His-tag antibodies exists 4, 5, 6, 7. Another standard technique to characterize and identify target protein containing fractions during and after purification is SDS-PAGE. A simple measurement of the UV-absorption is not specific enough to identify the target protein-containing fractions and can also be rather insensitive in case of proteins with a low tryptophane content and therefore a low extinction coefficient at 280 nm. After recombinant expression, the purification of His-tagged proteins using metal affinity chromatography is performed manually in most cases and the identification of the target protein-containing fractions is a tedious process. The observed K D values for this interaction are in the micromolar range and allow a highly specific purification of His-tagged proteins via metal-affinity chromatography 2, 3. The short hexa-His tag is one of the most commonly used protein tags and allows an easy and fast purification that is based on the strong affinity of histidine sequences to a nickel-complex (Ni-NTA). These amino acid sequences can then represent epitopes for specific binding partners like antibodies. Here, additional amino acid sequences are usually added to the terminal ends of the desired protein. As detection and purification of these proteins is often a difficult process, epitope tags are highly versatile and regularly used tools for that purpose 1. It substantially reduces the workload for recombinant protein detection, especially when His-tag-protein-containing fractions in manual chromatographic purifications have to be identified.Ĭloning and the subsequent recombinant expression of proteins is state-of-the-art in molecular biology and commonly used for decades. In conclusion, this fast, sensitive, specific and affordable mix-and-measure assay provides a timesaving and user-friendly way to quantify recombinant protein expression. Additionally, we demonstrate a full compatibility of the developed assay to cell lysate and a correlation to detectable bands in a western blot application. The detection limit is comparable to those obtained in SDS-PAGE or Western Blot, which are used as standard methods for the characterization of His-tag protein expression. The assay recognizes both, N- and C-terminally tagged proteins. The assay has a total assay time of less than two minutes including sample preparation. The targeted His-tag protein in the sample displaces the donor-labeled peptide and leads to a concentration-dependent time-resolved fluorescence signal. The assay is based on Förster resonance energy transfer (FRET) between a lanthanide dye-labeled low-affinity His-peptide and an acceptor-labeled anti-His-tag antibody. Here, we present a fast mix-and-measure immunoassay for the specific semiquantitative detection of His-tagged proteins, for example in E. ![]()
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