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?His-tags, or polyhistidine tags, comprise a consecutive series of six to ten histidine residues. The most common his-tag is the 6xHis tag and has a ? molecular weight of 0.8kDa1. His tags are used for many recombinant proteins to facilitate purification, allowing researchers to extract a protein of interest from thousands of other proteins found in a cell or cell lysate. The small size of the 6x-His tag means there is a lower possibility for the tag to affect the functionality of the fusion protein, which can happen with larger tags. ??
Histidine forms coordination bonds with immobilized transition metal ions, and this property can be utilized for protein purification. His-tag protein purification is by a specialized form of affinity chromatography, called immobilized metal affinity chromatography (IMAC), where proteins or peptides are separated according to their affinity for metal ions immobilized to a solid chelating resin. During this process, a small His-tag is fused to either the N or C terminus of the target protein, enabling capture by nickel or cobalt ions coordinated on a variety of resins. The small size, low cost, and ease of use have made the His-tag the most popular affinity-tag available2. However, as His residues exist in mammalian and insect systems, there can be background binding. This can be avoided by washing in 5 – 10 mM imidazole, but this washing step may prematurely elute the protein of interest.
For more information on affinity tags, including the His-tag, see our guide for fusion tags.
??Our range of antibodies, proteins, and assay kits are validated for use in various applications, including ELISA, western blot, immunohistochemistry, immunoprecipitation, immunocytochemistry, chromatin immunoprecipitation (ChIP), and flow cytometry.
Immunoprecipitation of His/FLAG tagged PDCD5 using ab18184 at 1/100 dilution.
Lane 1: MW Ladder (from top 120, 85, 50, 34, 25, 19 kDa)
Lanes 2-5: Immunoprecipitation of His/FLAG tagged PDCD5 using ab18184 from mixture of ~50 ng purified His/FLAG tagged PDCD5 and 15 μL RRL in 300 μL buffer.
Lanes 6-7: Purified PDCD5 from E. coli with ~60-70 ng protein.
The lowest band in each lane is ~ 20 kDa and is PDCD5. The upper bands in lanes 2-5 are the heavy chain (~50 kDa, runs as doublet) and light chain (~25 or 26 kDa).
Lane 1 : MW Ladder (from top 120, 85, 50, 34, 25, 19 kDa)
Lanes 2-5 : Immunoprecipitation of His/FLAG tagged PDCD5 using ab18184 from mixture of ~50 ng purified His/FLAG tagged PDCD5 and 15 μL RRL in 300 μL buffer.
Lanes 6-7 : Purified PDCD5 from E. coli with ~60-70 ng protein.
Immunoprecipitation - Anti-6X His tag? antibody [HIS.H8] (ab18184)
This image is courtesy of an Abreview submitted by Mr Chris Tracy
Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) cells transfected with GFP-Myc-His vector expression construct labeling 6X His tag? with ab213204 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor? 488) (ab150077) secondary antibody at 1/1000 dilution (white). Confocal image showing positive staining on HEK-293T cells transfected with GFP-Myc-His vector expression construct. The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor? 594) (ab195889) (red) at 1/200 dilution.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor? 488) (ab150077) secondary antibody at 1/1000 dilution.
|Immunocytochemistry/ Immunofluorescence - Anti-6X His tag? antibody [EPR20547] - ChIP Grade (ab213204)|
|Anti-6X His tag? antibodies||Applications|
|Anti-6X His tag? antibody [HIS.H8] (ab18184)||ICC/IF, ELISA, Dot blot, WB, IP, EMSA|
|Anti-6X His tag? antibody (HRP) (ab1187)||ELISA, WB|
|Anti-6X His tag? antibody (ab9108)||IP, WB, IHC-P, ICC/IF, ELISA, ChIP, Flow Cyt|
|Anti-6X His tag? antibody (FITC) (ab1206)||ELISA, WB, Flow Cyt|
|Anti-6X His tag? antibody [EPR20547] (ab213204)||WB, ICC/IF, IP, Flow Cyt, ChIP, IHC-P|
|ELISA and Assay kits||Sensitivity Range||Detection|
|GST 6XHis-tag ELISA Kit (ab128573)||> 0.5 ng/ml||Colorimetric|
|6X His tag? peptide (ab14943)|