6x-His Tag Monoclonal Antibody (3D5), AP, Invitrogen™

Description

NOTE: R932-25 was previously sold under product #46-0284. When re-ordering, please use #R932-25. R932-25 detects 6x His tag (C-term) -His-His-His-His-His-His-COOH. Detects fusion proteins containing this (requires the free carboxyl group for detection (Lindner et al., 1997). This product contains enough material for 25 western blots. Western blot experiments with 100 ng of recombinant Positope™ control protein gave a detectable signal within 15 minutes using a chromogenic substrate. The Positope™ control protein is a 53 kDa recombinant protein that contains seven epitope tags, including His(C-term), HisG, c-myc, and V5. For Western blot, do not dilute the AP-conjugated antibody into PBS. Phosphate inhibits alkaline phosphatase. We recommend diluting the AP-conjugated antibody in Tris-Buffered Saline (TBS) containing 0.1% (v/v) Tween-20 and 1% (w/v) nonfat dry milk. If you use a different buffer for washing and blocking your blots, then dilute as described above with that buffer. You may use other blocking agents such as bovine serum albumin (BSA) or gelatin. All AP-conjugated antibodies have been tested in immunoblotting procedures. Low background was observed. No cross-reactivity has been observed in bacterial lysates.

The 6x His tag is a synthetic oligo peptide consisting of 6 consecutive histidine residues (HHHHHH). The His tag is commonly expressed as a tag at either N-or C-terminal regions of recombinant proteins to allow isolation or purification by immobilized metal affinity chromatography. Epitope tagging is a technique in which a known epitope is fused to a recombinant protein using genetic engineering. By choosing a particular epitope and recombinant protein combination, epitope tagging makes it possible to detect proteins for which no antibody is available. His epitope tagged proteins that contain a stretch of histidine residues at the carboxyl or amino terminus are used for purification of recombinant proteins by means of metal chelate chromatography due to metal binding properties of histidine clusters on His tagged proteins. For example, 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. His tag-specific antibodies are used to facilitate detection or coimmunopreciptation of tagged proteins.