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525322 PhosphoDetect™ Phosphotyrosine Detection Kit

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      OverviewA set of four different monoclonal antibodies specific for tyrosine phosphorylation sites. Recognition is dependent on phosphorylation and the surrounding amino acid motif. Reacts with tyrosine phosphorylated proteins in chicken, human, mouse, rat, Xenopus, yeast, and zebrafish. Phosphorylation patterns in cell extracts may differ when probed with different antibodies. Phosphotyrosine Molecular Weight Markers (Cat. No. 525324)
      Catalogue Number525322
      Brand Family Calbiochem®
      Product Information
      Kit contains25 μg of each of the phosphotyrosine antibodies including clones 2C8, 3B12, 9H8, and 16F4, and a data sheet.
      Quality LevelMQ100
      Key Applications Enzyme-Linked Immunosorbent Assay
      Immunoblotting (Western Blotting)
      Biological Information
      Physicochemical Information
      Materials Information
      Toxicological Information
      Safety Information according to GHS
      Safety Information
      R PhraseR: 20/21/22

      Harmful by inhalation, in contact with skin and if swallowed.
      S PhraseS: 36

      Wear suitable protective clothing.
      Product Usage Statements
      Storage and Shipping Information
      Ship Code Blue Ice Only
      Toxicity Harmful
      Storage -20°C
      Do not freeze Ok to freeze
      Packaging Information
      Transport Information
      Supplemental Information
      Kit contains25 μg of each of the phosphotyrosine antibodies including clones 2C8, 3B12, 9H8, and 16F4, and a data sheet.
      Global Trade Item Number
      Catalog Number GTIN
      525322-1KIT 04055977270709


      PhosphoDetect™ Phosphotyrosine Detection Kit SDS


      Safety Data Sheet (SDS) 

      PhosphoDetect™ Phosphotyrosine Detection Kit Certificates of Analysis

      TitleLot Number
      User Protocol

      Revision06-January-2010 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.
      Principles of the assayAntibodies 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.

      Therefore, Calbiochem® brand offers a set of four different monoclonal antibodies specific for tyrosine phosphorylation sites. The phosphorylation patterns in a given cell extract may differ when probed with individual antibodies due to sequence specificity. These antibodies are suitable for use in human, mouse, rat, chicken, frog, zebrafish, and yeast cell extracts. The antibodies are not suitable for use with bacterial cell lysates.
      Materials providedThis kit contains 25 µg of each of the following phosphotyrosine antibodies:

      Table 1: Kit Contents

      Reconstitute each antibody in 1 ml of distilled water at room temperature. Aliquot and store at -70°C. *Use BSA for membrane blocking and antibody dilutions, do not use milk/casein.

      Detailed protocolPositive Control

      The Calbiochem® Phosphotyrosine Detection Kit includes a positive control (phosphotyrosine molecular weight marker component no. KP3905) consisting of seven phosphotyrosine-modified proteins: galactosidase (114 kDa), phosphorylase A (94 kDa), conalbumin (78 kDa), BSA (67 kDa), ovalbumin (47 kDa), carbonic anhydrase (30 kDa), and soybean trypsin inhibitor (21 kDa).

      Reconstitute the positive control in 200 µl of water. After solubilization, add 500 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 on PVDF membranes, apply 20 µl/lane (mini gel) for colorimetric detection or 10 µl/lane (mini gel) for chemiluminescent detection. Do not use nitrocellulose membranes!

      Figure 1: Blot

      Detection of phosphotyrosine-containg proteins by immunoblotting. Sample: Whole cell lysates from A431 cells treated with pervanadate (all lanes). Primary antibody: Anti-Phosphotyrosine (2C8) Mouse mAb (Cat. No. 525293) (0.5 µg/ml), Anti-Phosphotyrosine Mouse mAb (3B12) (0.5 µg/ml), Anti-Phosphotyrosine Mouse mAb (9H8) (0.5 µg/ml), and Anti-Phosphotyrosine Mouse mAb (16F4) (0.5 µg/ml). Detection: chemiluminescence.

      Registered TrademarksCalbiochem® is a registered trademark of EMD Chemicals, Inc.
      Interactive Pathways™ is a trademark of EMD Chemicals, Inc.