Total cell lysate (15 µg protein) from 293E (lane 1) and 293E transfected with His-tag fused protein (lane 2) were resolved by electrophoresis (4-20% Tris-Glycine gel), transferred to nitrocellulose, and probed with 1:1000 diluted (0.5 µg/mL), 1:2500 diluted (0.2 µg/mL) and 1:10000 diluted (0.05 µg/mL) purified anti-His tag antibody (clone J095G46). Proteins were visualized by chemiluminescence detection using 1:3000 diluted HRP Goat anti-mouse IgG Antibody (Cat. No. 405306, upper). 1:2000 dilution of Direct-Blot™ HRP anti-GAPDH antibody (clone FF26A/F9, Cat. No. 649203) was used as a loading control (lower). Lane M: MW ladder.
Total cell lysate (15 µg protein) from 293E (lane 1) and 293E transfected with His-tag fused protein (lane 2) were resolved by electrophoresis (4-20% Tris-Glycine gel), transferred to nitrocellulose, and probed with 1:1000 diluted (0.5 µg/mL), 1:2500 diluted (0.2 µg/mL) and 1:10000 diluted (0.05 µg/mL) purified anti-His tag antibody (clone J095G46). Proteins were visualized by chemiluminescence detection using 1:3000 diluted HRP Goat anti-mouse IgG Antibody (Cat. No. 405306, upper). 1:2000 dilution of Direct-Blot™ HRP anti-GAPDH antibody (clone FF26A/F9, Cat. No. 649203) was used as a loading control (lower). Lane M: MW ladder.
HeLa cells transiently transfected with His tag fused protein or non-transfected were fixed with 100% methanol for 10 minutes and blocked with 5% FBS for 60 minutes. Then the cells were stained overnight at 4 degrees with (A) 1: 500 diluted (1 µg/ml) mouse IgG2a isotype control (Cat. No. 401501)or, anti-His tag antibody at (B) 1: 500 (1 µg/ml), (C) 1: 1667 (0.3 µg/ml), (D) 1: 5000 (0.1 µg/ml) dilution, followed by 1:250 diluted (2 µg/ml) Alexa Fluor® 488 (green) conjugated goat anti-mouse IgG (Cat. No. 405319) antibody for one hour at room temperature. Nuclei were counterstained with DAPI (blue). The image was captured with a 60X objective.
This antibody was raised against a linear 8XHis tag. It reacts with His-tagged protein with C, N or internal 6XHis tags or longer. It can be used for western blotting to detect linear His repeats (6XHis or more). It can also be used for ICFC and ICC staining of His-tagged proteins.
Each lot of this antibody is quality control tested by Western blotting. For Western blotting, the suggested use of this reagent is 1:1000-1:10000 diluted (0.05 - 0.5 µg/mL). For immunocytochemistry, a concentration range of 0.1 - 1.0 µg/mL (1:500-1:5000 dilution) is recommended. For flow cytometric staining, the suggested use of this reagent is ≤ 0.5 µg per million cells in 100 µL volume. It is recommended that the reagent be titrated for optimal performance for each application.
Application Notes
This antibody was raised against a linear 8X His tag. It reacts with proteins displaying repeats of 6 Histidines (His6) or more in either internal or C and N terminal regions. It is suitable for ICFC.
Clone J095G46 has been verified for immunocytochemistry (ICC).
Product Citations
Zhang S, et al. 2021. Front Oncol. 585457:11. PubMed
Sokal A, et al. 2021. Cell. 184(5):1201-1213.e14. PubMed
Alberts A, et al. 2020. Front Immunol. 11:596103. PubMed
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