Anti-Phosphohistidine (pHis) (C15-1317-767)

General description

Phosphorylation plays an important role in regulating protein activities and various cellular signaling events in cells. Limited by the tools available for phosphohistidine (pHis) detection, the majority of studies focus on serine, threonine, and tyrosine phosphorylations. Histidine phosphorylation can occur at either N1 (1-pHis) or N3 (3-pHis) of the imidazole ring. The development of stable phosphoryltriazolylalanine analogues of pHis (pTza and pPza) allows the generation of antibodies for studying both histidine N1 and N3 phosphorylations in signaling events. There is growing evidence implicating His kinases in cancer and tumor metastasis and the first metastasis suppressor gene identified is one of the two known mammalian His kinases, Nm23-H1 (also known as NME1, nucleoside diphosphate kinase, or NDPK-A). Nm23-H1/NME1 and the closely related Nm23-H2 (NME2/NDPK-B) catalyze the transfer of phosphate from ATP onto Nucleoside-diphosphates (NDPs) through a 1-pHis enzyme intermediate. Nm23-H1/-H2 also possess His kinase activity, transferring the phosphate from the active site pHis onto a His in a target protein. Metabolic enzymes such as phosphoglycerate mutase (PGAM), succinyl CoA synthase (SCS), and ATP citrate lyase (ACL) also use pHis as an enzyme intermediate. Unlike NME1/2, PGAM uses 3-pHis as an enzyme intermediate. In addition to eukaryotes, histidine phosphorylation is well documented in bacterial two-component signaling pathways involved in chemotaxis, although the phosphate is transferred from the pHis formed in the receptor/sensor protein to Asp residues of an acceptor response regulator protein, and the receptor/sensor protein essentially functions as an aspartate kinase.

Specificity

Target modification is not species-specific.

This rabbit polyclonal antibody reacted with pHis- and pPza-, but not His-, pTyr-, pSer-, or pThr-, conjugated BSA. Specificity testing using N1-pHis analog showed minimal cross-reactivity.

Immunogen

KLH-conjugated non-hydrolyzable phosphohistidine analogue 4-phosphopyrazol-2-yl alanine (pPza).

Application

Detect histidine phosphorylation using this rabbit polyclonal Anti-Phosphohistidine (pHis) , Cat. No. ABS1670, validated for use in Dot Blot, ELISA, Immunoprecipitation, and Western Blotting.

Dot Blot Analysis: A 1:1,200 dilution from a representative lot detected 5 µg of pHis- and pPza-, but not His-, pTyr-, pSer-, or pThr-, conjugated BSA (Courtesy of Bezaleel Mambwe, Richmond Muimo and RFW Jackson, Department of Infection, Immunity and Cardiovascular Disease/ Department of Chemistry, University of Sheffield, UK).

Western Blotting Analysis: A 1:120 dilution from a representative lot detected proteins with histidine phosphorylation in sheep trachea cytosolic extract and human cell lysates, including 16HBE14o-, HEK293T, THP-1, and THP-1-derived macrophages (Courtesy of Bezaleel Mambwe, Richmond Muimo and RFW Jackson, Department of Infection, Immunity and Cardiovascular Disease/ Department of Chemistry, University of Sheffield, UK).

ELISA Analysis: A representative lot detected pHis- and pPza-, but not pTyr-, pSer-, pThr-, conjugated BSA or unconjugated BSA (Lilley, M., et al. (2015). Chem. Commun. (Camb). 51(34):7305-7308).

Immunoprecipitation Analysis: A representative lot immunoprecipitated proteins with histidine phosphorylation from ovine airway epithelia extract (Lilley, M., et al. (2015). Chem. Commun. (Camb). 51(34):7305-7308).

Western Blotting Analysis: A representative lot detected histidine phosphorylated proteins in 16HBE14o- human bronchial epithelial cell lysate and in ovine airway epithelia extract. Acid (0.1 M HCl or 0.4 M acetic acid/0.1 M hydroxylamine), but not alkaline (0.1 M NaOH), treatment of the lysates abolished targets bands detection (Lilley, M., et al. (2015). Chem. Commun. (Camb). 51(34):7305-7308).

Western Blotting Analysis: A representative lot detected histidine phosphorylation of immunoprecipitated NDPK-A/B from ovine airway epithelia extract, as well as G -R/M from 16HBE14o- human bronchial epithelial cell lysate (Lilley, M., et al. (2015). Chem. Commun. (Camb). 51(34):7305-7308).

Note: DO NOT HEAT SAMPLES prior to phosphohistidine detection. Histidine phosphorylation is heat and acid labile. To generate negative control for specificity test, an aliquot of sample can be heated at 95ºC for 10-15 minutes to reverse histidine phosphorylation. Alternatively, an aliquot of sample can be incubated under acidified pH at 37ºC for 15 minunites to reduce histidine phosphorylation. Acidify each 100 µL sample with 25 µL of 1 M HCl before the incubation, then neutralize with 25 µL of 1 M NaOH prior to phosphohistidine detection.

Research Category
Signaling

Quality

Evaluated by Western Blotting in HEK293 cell lysate.

Western Blotting Analysis: A 1:200 dilution of this antibody detected histidine-phosphorylated proteins in 10 µg of HEK293 cell lysate.

Target description

Variable depending on the histidine-phosphorylated proteins.

Physical form

Affinity purified.

Purified rabbit polyclonal antibody in buffer containing 100 mM glycine pH 2.2, 100 mM Tris pH 10 with 0.05% sodium azide.

Storage and Stability

Stable for 1 year at 2-8°C from date of receipt.

Other Notes

Concentration: Please refer to lot specific datasheet.

Disclaimer

Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.

biological source: rabbit. Quality Level: 100. antibody form: affinity isolated antibody. antibody product type: primary antibodies. clone: polyclonal. purified by: affinity chromatography. species reactivity: all, human, sheep, mouse. technique(s): ELISA: suitable, dot blot: suitable, immunoprecipitation (IP): suitable, western blot: suitable. shipped in: ambient. target post-translational modification: phosphorylation (pHis ). Gene Information: human ... PHPT1(29085). Storage Class Code: 12 - Non Combustible Liquids. WGK: WGK 1. Flash Point(F): Not applicable. Flash Point(C): Not applicable.