"I have used a wide variety of secondaries and Jackson ImmunoResearch has consistently been the best. The fluorophores are bright and stable and their selective (x reactivity removed) secondaries have always shown species specificity in multiple labeling."
PolyHistidine Tags (His Tags) are a popular addition to recombinant proteins to facilitate protein purification and detection. His Tags are commonly used in preference to other tags due to the availability of inexpensive purification resins such as NiNTA. Anti-His Tag antibodies enable the detection of the tagged proteins allowing screening of expression. The poly-His Tag is usually formed of 6 histidine residues but may be between three and ten, usually at the C- or N-terminus of the recombinant protein. Here we demonstrate the utility of Jackson ImmunoResearch’s Anti-His Tag antibodies for the detection of His-Tagged proteins by both Western blotting and ELISA.
Confidently screen His-Tagged proteins in cell lysates by Western blot
Protein expression screening of His-Tagged recombinant proteins (constructs) from cell lysates is a popular application of Anti-His Tag antibodies. Anti-His Tag antibodies allow the detection of both carboxy- and amino- terminally His-Tagged proteins. Figure 1 demonstrates the detection of a recombinant His-Tagged protein from mammalian, insect, and bacterial cell lysates.
Use conjugated Anti-His Tag antibodies directly for single-step Western blotting
Jackson ImmunoResearch’s Anti-His Tag antibodies can be used directly using a Horseradish Peroxidase conjugate. Figures 2 and 3 demonstrate that Jackson ImmunoResearch’s HRP Rabbit Anti-His Tag antibody can be used across a range of dilutions and that sensitive detection of different His-Tagged proteins can be achieved. The sensitivity of the Western blot assay can be enhanced by performing the assay indirectly, using a conjugated secondary antibody to achieve increased signal shown in the following section.
Enhance sensitivity by using Jackson ImmunoResearch’s Anti-His Tag antibody in combination with Anti-Rabbit conjugates
Signal enhancement can be achieved by indirect detection of the His Tag using an Anti-His Tag antibody followed by a conjugated Anti-Rabbit antibody such as an HRP Goat Anti-Rabbit secondary antibody. Figure 4 shows that sensitivity can be improved by using the indirect two-step method (B) in comparison to direct detection (A).
Anti-His Tag antibody for ELISA
Figure 5 shows the variation in binding characteristics of a range of recombinant His-Tagged Alpaca VHH antibodies isolated from an activity screen against antigen X and analyzed by ELISA. Variations in binding affinity of the different nanobodies can clearly be discerned and lead candidates selected.
Screening ELISA using His Tag capture
The His-Tag offers another method of protein capture that may be useful when working with proteins subject to conformational sensitivity, allowing the protein to be elevated above the plate surface making more epitopes accessible to immunoreagents. Another application for the Anti-His Tag antibody is in situations where there is limited target protein-specific antibody availability, but the assay requires two different antibodies to generate the sandwich format. By capturing with the His Tag, target protein-specific antibodies can be used for detection in complex with conjugated species-specific secondary antibodies, allowing optimal signal amplification. His Tag capture also provides antibody-mediated, rapid and consistent binding of antigen to the ELISA plate.
* We recommend using a detection antibody cross-adsorbed against rabbit serum proteins when using a rabbit antibody during the capture step to avoid background. Jackson ImmunoResearch antibodies, including anti-human, are available with minimal cross-reactivity to a variety of hosts, allowing you to abrogate background from multiple host species.
1LIA CRYSTAL STRUCTURE OF R-PHYCOERYTHRIN FROM POLYSIPHONIA AT 2.8 A RESOLUTION from PDB Chang, W. R., Jiang, T., Wan, Z. L., Zhang, J. P., Yang, Z. X., & Liang, D. C. (1996). Crystal structure of R-phycoerythrin from Polysiphonia urceolata at 2.8 A resolution. Journal of molecular biology, 262(5), 721–731. https://doi.org/10.1006/jmbi.1996.0547 Image generated using Molsoft ICM browser
HIS-TAG® is a trademark of EMD Biosciences, Inc.
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