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Western blot analysis of Histidine tag (6X His) was performed by loading 20 µg of whole cell extracts of untransfected HEK-293 and HEK-293 transiently overexpressing His-IGFBP6 using Novex® NuPAGE® 4-12% Bis-Tris gel (NP0321BOX), Xcell SureLock™ Electrophoresis system (EI0002), Novex Sharp Pre-stained Protein Standard (LC5800), and iBlot® Dry Blotting System (IB21001). Proteins were transferred to a nitrocellulose membrane and blocked with 5% skim milk for 1 hour at room temperature. His-IGFBP6 was detected at ~31 kDa using Histidine (C- term) Tag (6XHis) - HRP Mouse Monoclonal Antibody (R93125) at a 1:3000 dilution in 2.5% skim milk at 4°C overnight on a rocking platform. Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (WP20005).
|Tested species reactivity||Tag|
|Host / Isotype||Mouse / IgG2b|
|Immunogen||6x His synthetic peptide|
|Storage Conditions||Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.|
|Tested Applications||Dilution *|
|Western Blot (WB)||1:1000-1:5000|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
This antibody can be used to detect expression of C-terminal His fusion proteins from bacterial, insect, and mammalian cells.
This product contains enough material for 25 Western blots. It is functionally tested using 100ng of an E. coli expressed fusion protein containing a C-terminal polyhistidine (6xHis) epitope. Chemiluminescent detection should be performed using a 1 minute exposure. 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.
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.
His Tag; Histidine Epitope Tag; Histidine Tag