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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) -AP Mouse Monoclonal Antibody (R93225) at 1:3000 dilution in 2.5% skim milk at 4°C overnight on a rocking platform and chemiluminescent detection was performed using AP Chemiluminescence technique (SLF1022).
|Tested species reactivity||Tag|
|Host / Isotype||Mouse / IgG2b|
|Immunogen||6x His synthetic peptide: C-term -His-His-His-His-His-His-COOH|
|Contains||0.01% sodium azide|
|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.
R932-25 detects 6x His tag (C-term) -His-His-His-His-His-His-COOH.
This product contains enough material for 25 Western blots. Western blot experiments with 100ng 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, 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.
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.