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MABS1352 Anti-N3-Phosphohistidine (3-pHis) Antibody, clone SC56-2

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      Key Specifications Table

      Species ReactivityKey ApplicationsHostFormatAntibody Type
      H, E. coliWB, DBRbPurifiedMonoclonal Antibody
      Catalogue NumberMABS1352
      DescriptionAnti-N3-Phosphohistidine (3-pHis) Antibody, clone SC56-2
      Alternate Names
      • N3-Phosphohistidine
      Background InformationPhosphorylation 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 peptides containing stable phosphoryltriazolylalanine analogues of 1-pHis and 3-pHis (1-pTza and 3-pTza) 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.
      Product Information
      PresentationPurified rabbit monoclonal antibody in buffer containing 0.1 M Tris-Glycine (pH 7.4), 150 mM NaCl with 0.05% sodium azide.
      Quality LevelMQ100
      ApplicationAnti-N3-Phosphohistidine (3-pHis) antibody, clone SC56-2 is an isomer-specific monoclonal Ab to specifically detect histidine phosphorylated at position N3. This purified mAb is backed by published data demonstrating performance in Western blotting and dot blot applications.
      Key Applications
      • Western Blotting
      • Dot Blot
      Application NotesDot Blot Analysis: A representative lot detected peptides containing 3-pTza phosphohistidine analogue, but not peptides containing only phosphorylated tyrosine. Clone SC56-2 is most reactive toward 3-pTza peptides based on NM23-H1/NME1 3-pHis118 and PGAM 3-pHis11 sequence, less reactive toward 3-pTza peptides based on ACLY 3-pHis760, histone H4 3-pHis18, or Kca3.1 3-pHis358 sequence, while being least reactive toward GNB1 3-pHis266-based 3-pTza peptide (Fuhs, S.R., et al. (2015). Cell. 162(1):198-210).

      Western Blotting Analysis: Clone SC56-2 hybridoma culture supernatant was employed for Western blotting analysis of heat-sensitive histidine N3-phosphorylation (3-pHis) of exogenously expressed human PGAM GST fusion protein in lysates from transformed E. coli (Fuhs, S.R., et al. (2015). Cell. 162(1):198-210).

      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 minutes 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.
      Biological Information
      ImmunogenKLH-conjugated library of random peptides containing non-hydrolyzable phosphohistidine analogue 3-pTza.
      EpitopeN3-phosphohistidine (3-pHis)
      ConcentrationPlease refer to lot specific datasheet.
      SpecificitySelectively detects proteins with histidine(s) phosphorylated at N3 of the imidazole ring (3-pHis), but not 1-pHis.
      Species Reactivity
      • Human
      • E. coli
      Species Reactivity NoteHuman, E.coli. Predicted to react with all species. Target modification is not species specific.
      Antibody TypeMonoclonal Antibody
      • Phosphorylation
      Purification MethodProtein A Purfied
      Molecular WeightVariable depending on the histidine-phosphorylated proteins.
      Physicochemical Information
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      Product Usage Statements
      Quality AssuranceEvaluated by Western Blotting of PGAM-catalyzed 2,3-DPG degradation reaction.

      Western Blotting Analysis: 0.52 µg/mL of this antibody detected recombinant human phosphoglycerate mutase (PGAM) with N3-phosphohistidine (3-pHis) in a 5 µg aliquot of PGAM-catalyzed 2,3-diphosphoglycerate (2,3-DPG) degradation reaction.
      Usage Statement
      • 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.
      Storage and Shipping Information
      Storage ConditionsStable for 1 year at 2-8°C from date of receipt.
      Packaging Information
      Material Size100 µL
      Transport Information
      Supplemental Information


      Anti-N3-Phosphohistidine (3-pHis) Antibody, clone SC56-2 SDS


      Safety Data Sheet (SDS) 

      Anti-N3-Phosphohistidine (3-pHis) Antibody, clone SC56-2 Certificates of Analysis

      TitleLot Number
      Anti-N3-Phosphohistidine (3-pHis), -2789413 2789413
      Anti-N3-Phosphohistidine (3-pHis), clone SC56-2 - 3172393 3172393
      Anti-N3-Phosphohistidine (3-pHis), clone SC56-2 - 3227881 3227881
      Anti-N3-Phosphohistidine (3-pHis), clone SC56-2 - 3257006 3257006
      Anti-N3-Phosphohistidine (3-pHis), clone SC56-2 - 3425376 3425376
      Anti-N3-Phosphohistidine (3-pHis), clone SC56-2 - 3460521 3460521
      Anti-N3-Phosphohistidine (3-pHis), clone SC56-2 - 3502126 3502126
      Anti-N3-Phosphohistidine (3-pHis), clone SC56-2 - 3598000 3598000
      Anti-N3-Phosphohistidine (3-pHis), clone SC56-2 - 3720066 3720066
      Anti-N3-Phosphohistidine (3-pHis), clone SC56-2 - 3764738 3764738


      Reference overviewPub Med ID
      Monoclonal 1- and 3-Phosphohistidine Antibodies: New Tools to Study Histidine Phosphorylation.
      Fuhs, SR; Meisenhelder, J; Aslanian, A; Ma, L; Zagorska, A; Stankova, M; Binnie, A; Al-Obeidi, F; Mauger, J; Lemke, G; Yates, JR; Hunter, T
      Cell  162  198-210  2015

      Show Abstract
      26140597 26140597

      Technical Info

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