Key features and details
- Sheep polyclonal to phosphohistidine
- Suitable for: WB, IP, Competitive ELISA
- Reacts with: Species independent
- Isotype: IgG
Product nameAnti-phosphohistidine antibody
DescriptionSheep polyclonal to phosphohistidine
Tested applicationsSuitable for: WB, IP, Competitive ELISAmore details
Species reactivityReacts with: Species independent
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.
- 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.
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.
Reproducibility is key to advancing scientific discovery and accelerating scientists’ next breakthrough.
Abcam is leading the way with our range of recombinant antibodies, knockout-validated antibodies and knockout cell lines, all of which support improved reproducibility.
We are also planning to innovate the way in which we present recommended applications and species on our product datasheets, so that only applications & species that have been tested in our own labs, our suppliers or by selected trusted collaborators are covered by our Abpromise™ guarantee.
In preparation for this, we have started to update the applications & species that this product is Abpromise guaranteed for.
We are also updating the applications & species that this product has been “predicted to work with,” however this information is not covered by our Abpromise guarantee.
Applications & species from publications and Abreviews that have not been tested in our own labs or in those of our suppliers are not covered by the Abpromise guarantee.
Please check that this product meets your needs before purchasing. If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, as well as customer reviews and Q&As.
Storage instructionsShipped 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 bufferpH: 7.40
Preservative: 0.05% Sodium azide
Constituents: Tris glycine, 0.87% Sodium chloride
Concentration information loading...
Our Abpromise guarantee covers the use of ab231709 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IP||Use at an assay dependent concentration.|
|Competitive ELISA||Use at an assay dependent concentration.|
- tele-phosphohistidine antibody
Competitive ELISA of ab231709 against phosphorylated amino acids and His, using τ-pHis conjugated to BSA.
Anti-phosphohistidine antibody (ab231709) at 1/4000 dilution + Human bronchial epithelial cell lysate at 100 µg
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.
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
ab231709 has not yet been referenced specifically in any publications.