Recombinant
RabMAb

Recombinant Anti-Phosphothreonine antibody [EPR22006-23] (ab218195)

Overview

  • Product name

    Anti-Phosphothreonine antibody [EPR22006-23]
    See all Phosphothreonine primary antibodies
  • Description

    Rabbit monoclonal [EPR22006-23] to Phosphothreonine
  • Host species

    Rabbit
  • Tested applications

    Suitable for: WB, ELISAmore details
    Unsuitable for: Flow Cyt,ICC/IF or IP
  • Species reactivity

    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide within Phosphothreonine. The exact sequence is proprietary.

  • Positive control

    • WB: Jurkat, C6 and NIH/3T3, treated with Calyculin A, whole cell lysate. Jurkat, treated with pervanadate, whole cell lysate.
  • General notes

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab218195 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB 1/1000.
ELISA 1/10000.
  • Application notes
    Is unsuitable for Flow Cyt,ICC/IF or IP.
  • Target

    • Relevance

      Phosphorylation of threonine residues is associated with many growth factors and oncogene protein kinases, and is important for cell signaling in activation, proliferation and differentiation. Protein phosphorylation and dephosphorylation are basic mechanisms for the modification of protein function in eukaryotic cells. Phosphorylation is a rare post-translational event in normal tissue, however, the abundance of phosphorylated cellular proteins increases several fold following various activation processes which are mediated through phosphotyrosine, phosphoserine or phosphothreonine (p-tyr/p-ser/p-thr). Many signal transduction pathways, such as the EGF, PDGF and insulin receptor systems, contain tyr/ser/thr kinase which phosphorylate specific tyr/ser/thr residues upon binding of ligands to their receptors. T cell antigen receptor complex or the receptors for some hemopoietic growth factors may stimulate these phosphorylation associated kinases, and cells transformed by viral oncogenes contain elevated levels of phosphorylated tyr/ser/thr. An understanding of transformation by oncogenes and mitogenic processes of growth factors depends on the identification of their substrate and a subsequent determination of how phosphorylation affects their properties. Studies on the role of phosphorylated proteins have been hampered by their low abundance and the problem of distinguishing the various types of phosphorylated proteins. The most common procedure is to label intact cells or small tissue fragments with 32P and subsequently to isolate 32P labeled proteins by conventional biochemical methods. In order to identify the specific amino acids that undergo phosphorylation, additional long and tedious procedures for phosphoamino acid analysis are required. Immunoblotting of cellular proteins with antibodies directed against phosphoamino acids is advantageous as it does not involve 32P labeling, and can therefore be employed to monitor alterations in phosphorylation of specific proteins as they occur in intact organs or the whole animal. Indeed, mono and polyclonal antibodies directed against phosphorylated residues have been generated and found useful as analytical and preparative tools because they enable the rapid identification, quantification and immunoaffinity isolation of phosphorylated cellular proteins.

    Images

    • All lanes : Anti-Phosphothreonine antibody [EPR22006-23] (ab218195) at 1/1000 dilution

      Lane 1 : Untreated Jurkat (human t cell leukemia T lymphocyte) whole cell lysate (Untreated membrane)
      Lane 2 : Jurkat treated with 0.1 µM Calyculin A for 45 minutes, whole cell lysate (Untreated membrane)
      Lane 3 : Jurkat treated with 0.1 µM Calyculin A for 45 minutes, whole cell lysate (Phosphatase treated membrane)

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

      Exposure time: 3 minutes


      Blocking/Dilution buffer: 5% NFDM/TBST.

      Observed MW: Multiple smeared bands.

    • All lanes : Anti-Phosphothreonine antibody [EPR22006-23] (ab218195) at 1/1000 dilution

      Lane 1 : Untreated NIH/3T3 (mouse embryonic fibroblast) whole cell lysate
      Lane 2 : NIH/3T3 treated with 0.1 uM Calyculin A for 15 minutes, whole cell lysate

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

      Exposure time: 59 seconds


      Blocking/Dilution buffer: 5% NFDM/TBST.

      Observed MW: Multiple smeared bands.

    • All lanes : Anti-Phosphothreonine antibody [EPR22006-23] (ab218195) at 1/1000 dilution

      Lane 1 : Untreated C6 (rat glial tumor glial cell) whole cell lysate
      Lane 2 : C6 treated with 0.1 uM Calyculin A for 30 minutes, whole cell lysate

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

      Exposure time: 3 minutes


      Blocking/Dilution buffer: 5% NFDM/TBST.

      Observed MW: Multiple smeared bands.

    • All lanes : Anti-Phosphothreonine antibody [EPR22006-23] (ab218195) at 1/1000 dilution

      Lane 1 : Untreated Jurkat (human t cell leukemia t lymphocyte) whole cell lysate at 20 µg
      Lane 2 : Jurkat treated with 50 mM pervanadate for 5 minutes, whole cell lysate at 10 µg

      Secondary
      All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

      Exposure time: 6 seconds


      No difference in the signal detected by ab218195 upon pervanadate (a protein tyrosine phosphatase inhibitor) treatment.

      Blocking/Dilution buffer: 5% NFDM/TBST.

    • ELISA - Anti-Phosphothreonine antibody [EPR22006-23] (ab218195).

      Each point in the Y axis represents one peptide of the corresponding protein used in ELISA test. The red asterisk indicates a phosphorylated amino acid site. The X axis is the signal to noise (S/N) ratio calculated by the signal intensity of a phospho peptide and its non-phospho paralog.

      Antigens used at 1 µg/ml: TWIST1(pY107); TP73(pY99); SQSTM1 (pS403); NFKB (pS337); Phosphoserine; Phosphothreonine; ADD1(pT445); EIF2AK3(pT982); AURKB(pT232); AKT1(pT308).

      Primary antibody ab218195 used at a 1/10,000 dilution.

      Secondary antibody Alkaline Phosphatase AffiniPure Goat Anti-Rabbit IgG (H+L) used at a 1/2,500 dilution.

    References

    ab218195 has not yet been referenced specifically in any publications.

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