HeLa cells were transfected with (B-D) or without (A) His-tagged cytoplasmic plasmid. Cells were fixed with 4% paraformaldehyde (PFA) for 15 minutes, permeabilized with 0.5% Triton X-100 for 3 minutes, and blocked with 5% FBS for 60 minutes. Then the cells were intracellularly stained with (A) Alexa Fluor® 647 Mouse IgG1, κ isotype control antibody (Negative, Cat. No. 400136) or (B-D) Alexa Fluor® 647 Anti-6-His antibody (clone 6-His) overnight at 4°C. Nuclei were counterstained with DAPI (blue, Cat. No. 422801). The image was captured with a 60X objective using KEYENCE BZ-X700 fluorescence microscope. Exposure time (Seconds) for (A-D) is 1/35. Concentrations for (A-B) is 2.5 µg/mL (1:200 dilution), (C) is 1 µg/mL (1:500 dilution) and (D) is 0.5 µg/mL (1:1000 dilution).
HeLa cells were transfected with (B-D) or without (A) His-tagged cytoplasmic plasmid. Cells were fixed with 4% paraformaldehyde (PFA) for 15 minutes, permeabilized with 0.5% Triton X-100 for 3 minutes, and blocked with 5% FBS for 60 minutes. Then the cells were intracellularly stained with (A) Alexa Fluor® 647 Mouse IgG1, κ isotype control antibody (Negative, Cat. No. 400136) or (B-D) Alexa Fluor® 647 Anti-6-His antibody (clone 6-His) overnight at 4°C. Nuclei were counterstained with DAPI (blue, Cat. No. 422801). The image was captured with a 60X objective using KEYENCE BZ-X700 fluorescence microscope. Exposure time (Seconds) for (A-D) is 1/35. Concentrations for (A-B) is 2.5 µg/mL (1:200 dilution), (C) is 1 µg/mL (1:500 dilution) and (D) is 0.5 µg/mL (1:1000 dilution).
His tags are commonly used as a method of tagging proteins for detection or purification. This antibody is raised again a polyhistidine tag (6-His or hexa His-tag).
Each lot of this antibody is quality control tested by immunocytochemistry. For immunocytochemistry, a concentration range of 0.5 - 2.5 μg/mL (1:200-1:1,000 dilution) is recommended. It is recommended that the reagent be titrated for optimal performance for each application.
* Alexa Fluor® 647 has a maximum emission of 668 nm when it is excited at 633 nm / 635 nm.
Alexa Fluor® and Pacific Blue™ are trademarks of Life Technologies Corporation.
The sequence of the protein flanking the poly His tag does not influence antibody binding. This antibody was subsequently purified via a protein-G column.
Application References
(PubMed link indicates BioLegend citation)
Deinert K, et al. 2001. J. Biol. Chem. 276:6000. (WB)
Kojima H, et al. 2001. J. Biol. Chem.(22)276:19238. (ICC) PubMed
Lapinski PE, et al. 2000. J. Biol. Chem. 275:6831. (WB)
Bartsch D, et al. 1998. Cell. 95:211. (ICC)
Fuentes EJ, et al. 2003. J Biol Chem. 278:2118. PubMed
Montegna EA, et al. 2012 PLoS One.7:31156. (WB) PubMed
Huang L, et al. 2013. Nat Biotechnol. 31:450. (WB) PubMed
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