Anti-phosphohistidine antibody (ab231709)
Key features and details
- Sheep polyclonal to phosphohistidine
- Suitable for: WB, IP, Competitive ELISA
- Reacts with: Species independent
- Isotype: IgG
Overview
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Product name
Anti-phosphohistidine antibody -
Description
Sheep polyclonal to phosphohistidine -
Host species
Sheep -
Tested Applications & Species
See all applications and species dataApplication Species IP Species independentWB Species independent
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Immunogen
Other Immunogen Type. A non-hydrolysable analogue of phosphohistidine, 1-(2-aminoethyl)pyrazol-4-ylphosphonic acid, conjugated to keyhole limpet hemocyanin (KLH) via a glutaraldehyde linkage.
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Positive control
- Competitive ELISA: Phosphorylated amino acids and His, using t-pHis conjugated to BSA. WB: Human bronchial epithelial cells. IP: pHis proteins from human bronchial epithelial cells.
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General notes
It is recommended the antibody is centrifuged at 11200 RCF at 4°C for 10 minutes before use and supernatant used for analysis.
The following buffers and conditions for the Western blot are suggested:
Lysis buffer: 150 mM sodium chloride, 0.5 % sodium deoxycholate, 1% Triton X-100, 0.1 % sodium dodecyl sulfate, 10 mM sodium fluoride, 5 mM sodium orthovanadate, 10 mM sodium pyrophosphate, protease inhibitor cocktail, 50 mM tris base, pH 9.0.Denature samples in 5x loading buffer with; 10 % lithium dodecyl sulphate, 40% glycerol, 0.02% bromophenol blue, 50 mM ethylenediaminetetraacetic acid disodium salt dihydrate, 500 mM dithiothreitol, 300 mM tris base, pH 8.8 for 30 min at RT (do not heat).
Block in; 10 mM tris-hydrochloride, 165 mM sodium chloride, pH 8.0, 0.05 % (v/v), Tween 20, 0.2 % (v/v) freshwater fish gelatin at RT for 1 hr.
As a control phosphohistidine can be reduced/abolished by reducing the pH (pH 2-4) in denaturing buffer and then heating between 60-90 ºC for > 30 min.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle. -
Storage buffer
pH: 7.40
Preservative: 0.05% Sodium azide
Constituents: Tris glycine, 0.87% Sodium chloride -
Concentration information loading... -
Purity
Affinity purified -
Clonality
Polyclonal -
Isotype
IgG
Images
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Competitive ELISA - Anti-phosphohistidine antibody (ab231709)
Competitive ELISA of ab231709 against phosphorylated amino acids and His, using τ-pHis conjugated to BSA.
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Western blot - Anti-phosphohistidine antibody (ab231709)Anti-phosphohistidine antibody (ab231709) at 1/4000 dilution + Human bronchial epithelial cell lysate at 100 µg
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Immunoprecipitation - Anti-phosphohistidine antibody (ab231709)Immunoprecipitation of pHis proteins from human bronchial epithelial cells using ab231709. Western blot of immunoprecipitates using ab231709 at 1/4000 dilution.
pHis antibodies are immobilised using protein G sepharose. Elution refers to batch extraction with competitor. pHis antibody used to probe proteins on PVDF membrane.
Right pair of images: pHis is known to be acid labile: Acid treatment (pH 7, 60ºC 30 minutes) of lysate before IP.