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525288 PhosphoDetect™ Phosphothreonine Detection Kit

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      OverviewA set of three different monoclonal antibodies specific for threonine phosphorylation sites. Recognition is dependent on phosphorylation and the surrounding amino acid motif. Reacts with threonine phosphorylated proteins in Arabidopsis, bacteria, chicken, human, maize, mouse, rat, tobacco, Xenopus, yeast, and zebrafish. Phosphorylation patterns in cell extracts may differ when probed with different antibodies.
      Catalogue Number525288
      Brand Family Calbiochem®
      Product Information
      Kit contains25 μg of each of the Phosphothreonine Antibodies including clones 1E11, 4D11, and 14B3, Rabbit Muscle Positive Control, and a data sheet.
      Quality LevelMQ100
      Key Applications Enzyme-Linked Immunosorbent Assay
      Immunoblotting (Western Blotting)
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      Physicochemical Information
      Materials Information
      Toxicological Information
      Safety Information according to GHS
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      Product Usage Statements
      Storage and Shipping Information
      Ship Code Blue Ice Only
      Toxicity Standard Handling
      Storage -20°C
      Do not freeze Ok to freeze
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      Supplemental Information
      Kit contains25 μg of each of the Phosphothreonine Antibodies including clones 1E11, 4D11, and 14B3, Rabbit Muscle Positive Control, and a data sheet.
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      Catalog Number GTIN


      PhosphoDetect™ Phosphothreonine Detection Kit Certificates of Analysis

      TitleLot Number
      User Protocol

      Revision01-April-2008 RFH
      BackgroundProtein phosphorylation is the principal indicator of the activation of signaling pathways. The binding of a growth factor to its specific receptor catalyzes a complex cascade of intracellular signaling events, characterized by altering the phosphorylation of tyrosine, serine, or threonine residues of target proteins. The phosphorylation state is tightly regulated by the actions of protein kinases, which transfer a phosphate group from donor substrates such as ATP or GTP to serine, threonine, or tyrosine residues, and protein phosphatases, which dephosphorylate phosphorylated proteins, thereby restoring the particular phosphorylation system to its basal stage. Most kinases are either serine/threonine-specific or tyrosine-specific, while some kinases can phosphorylate all 3 types of hydroxy-bearing amino acids. Radioactive kinase assays utilizing 32P-γ-ATP as the donor substrate have been frequently employed to assess kinase activity, since the method is sensitive, convenient, and easily quantified. Recently, however, the generation of phosphoprotein-specific antibodies has advanced the development of non-radioactive kinase activity detection methods to comparable sensitivity as radioactive methods. Antibodies recognizing phosphorylated epitopes recognize the phosphorylated amino acid in the context of the surrounding amino acid sequence. Recognition, therefore, depends on 2 criteria: 1) phosphorylation and 2) the surrounding amino acid motif. If one of the two criteria is not fulfilled, the antibody will not detect the phosphorylation site. Since the surrounding amino acid sequence varies between phosphorylation sites, certain proteins though phosphorylated may not be detected by a given antibody.
      Principles of the assaySet of three different monoclonal antibodies specific for threonine phosphorylation sites. The phosphorylation patterns in a given cell extract may differ when probed with individual antibodies due to sequence specificity.
      Materials provided 25 µg of each of the following phosphothreonine antibodies:

      Table 1: Kit Contents

      *Use BSA for membrane blocking and antibody dilutions, do not use milk/casein.

      • Rabbit Muscle Positive Control: phosphoproteins purified from rabbit muscle by affinity chromatography using Fe3+-IDA-Sepharose; lyophilized from 20 mM Na₂HPO₄, 10 mM NaF, and 0.1% SDS.
      Reagent preparation• Antibodies: These antibodies were purified from serum-free cell culture supernatant by thiophilic adsorption and size exclusion chromatography. They are provided in lyophilized form, each lyophilized from 1 ml of 2x PBS containing 0.1% sodium azide and a small amount of PEG and sucrose. They should be reconstituted with 1 ml of sterile distilled water (15 min at room temperature). Following reconstitution, aliquot and freeze at -70°C. AVOID REPEATED FREEZE/THAW CYCLES. The reconstituted antibodies are stable for up to 1 year at -70°C. Before use, thaw the aliquot at 37°C. • Rabbit Muscle Positive Control: Reconstitute the positive control in 150 µl of water. After complete solubilization, add 150 µl of SDS-PAGE sample buffer and incubate at 90°C for 5 min. Aliquot and store at -20°C. Avoid freeze/thaw cycles. For immunoblotting, apply 20 µl/lane (mini gel) for colorimetric detection or 5 µl/lane (mini gel) for chemiluminescent detection.
      Example data

      Figure 1: Example Blot

      Detection of phosphothreonine-containing proteins by immunoblotting. Sample: Extract from rabbit muscle. Primary antibody: Anti-Phosphotheonine (14B3) (Mouse) (1 µg/ml) (Cat. No. 525286) (1 µg/ml). Anti-Phosphothreonine (1E11) (Mouse) (Cat. No. 525287) (1 µg/ml), and Anti-Phosphothreonine (4D11) (1 µg/ml). Detection: chemiluminescence.

      SpecificitySuitable for use in human, mouse, rat, chicken, frog, zebrafish, yeast, maize, tobacco, and bacterial cell extracts.
      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
      Interactive Pathways™ is a trademark of EMD Chemicals, Inc.