NOTE: R941-25 was previously sold under product #46-1009. When re-ordering, please use #R941-25.
This antibody is designed to specifically detect recombinant proteins containing the seven amino acid 6xHis-Gly epitope. This epitope is present in many expression vectors encoding an N-terminal 6xHis tag. The HisG antibody recognizes the following sequence: -His-His-His-His-His-His-Gly and can be used to detect expression of fusion proteins from bacterial, insect, and mammalian cells.
Anti-HisG-HRP Antibody was prepared by crosslinking the Anti-HisG Antibody with horseradish peroxidase using glutaraldehyde. It has been tested in immunoblotting and ELISA procedures. It was tested against purified Positope™ control protein (5 ng). The Positope™ control protein is a 53 kDa recombinant protein that contains seven epitope tags, including His(C-term), HisG, c-Myc, and V5. Low background was observed using chemiluminescent or alkaline phosphatase reagents for detection. In western blot experiments with purified protein, 50 ng (for Anti-HisG-HRP Antibody) of the Positope protein gave a detectable signal.
For western blot, dilute in PBS containing 0.05% Tween-20 and 5% nonfat, dry milk (PBSTM). Using chemiluminescence as the detection method, no cross-reactivity has been observed in bacterial lysates. In mammalian lysates, a few cross-reactive proteins have been observed upon overexposure of blots. If you use alkaline phosphatase-conjugated secondary antibody, do not use PBS. Phosphate inhibits alkaline phosphatase. Use Tris-Buffered Saline (TBS) instead. Be sure to wash the western blot or microtiter wells before adding the horseradish peroxidase-conjugated secondary antibody. Azide will inhibit HRP activity. This product contains enough material for 25 western blots; assumes 10 mL buffer per western blot.
Epitope tags provide a method to localize gene products in a variety of cell types, study the topology of proteins and protein complexes, identify associated proteins, and characterize newly identified, low abundance or poorly immunogenic proteins when protein-specific antibodies are not available. Expression of recombinant proteins in E. coli as a fusion protein with neighboring histidine residues is one of the most popular methods of epitope tags. The affinity of the histidine-tag motif to Ni2+ by chelation is strong and selective enough to enable purification of the protein to homogeneity by affinity chromatography on a Ni2 +-NTA adsorbent.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.
Protein Aliases: 6xHis-Gly; 6xHis-Glycine; His G; His Tag; Histidine Epitope Tag; Histidine Tag