Overview

  • Product name
    Anti-Phosphothreonine antibody (Biotin)
    See all Phosphothreonine primary antibodies
  • Description
    Rabbit polyclonal to Phosphothreonine (Biotin)
  • Host species
    Rabbit
  • Conjugation
    Biotin
  • Specificity
    Reacts with free phosphothreonine but does not react with phosphoserine, threonine or phosphotyrosine.
  • Tested applications
    Suitable for: WB, IP, ELISAmore details
  • Species reactivity
    Reacts with: Species independent
  • Immunogen

    Chemical/ Small Molecule conjugated to KLH.

  • Positive control
    • Use mouse brain extract for immunoblotting. Use synthetic phosphopeptide (on threonine) for ELISA.

Properties

Applications

Our Abpromise guarantee covers the use of ab9340 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
IP
ELISA
  • Application notes
    ELISA(kinase assay): Use at 0.5 µg/mL
    Western blot: Use at 4µg/mL
    IP: Use at 10 µg/250 µg protein sample
    Will detect 100 ng of phosvitin in Western Blots and 0.5 ng of phosvitin with ELISA.
    Can be used for non-radioactive protein kinase assay (ELISA) using biotinylated peptide substrate and immunoblotting of abundant phosphoproteins.
    It is not recommended for immunoblotting of trace cellular phosphoproteins. Acetone precipitation of the protein extract followed by SDS denaturation is recommended for successful immunoprecipitation.
  • 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.

    References

    This product has been referenced in:
    • Zhang C  et al. Nuclear accumulation of symplekin promotes cellular proliferation and dedifferentiation in an ERK1/2-dependent manner. Sci Rep 7:3769 (2017). WB ; Human . Read more (PubMed: 28630428) »
    See 1 Publication for this product

    Customer reviews and Q&As

    1-3 of 3 Abreviews or Q&A

    Answer

    The immunogen is phosphothreonine-KLH conjugates. The antibody is affinity-purified with phosphothrenonine and adsorbed by threonine on Agarose. The antibody recognized beter when the p-thre is next to glycine. Also, at the antiboy concentration that yield maximum OD, no apparent signal response to p-tyr.

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    Question

    Follow-up correspondance: -which antibody was used for the immunoprecipitation step(we have on anti-Ets-1 antibody but it has not been tested in IP). I used an antibody from Santa Cruz for Ets-1 which has been tested for IP and which I have used before successfully for IP -Has the antibody purchased been tested in IP? See above answer - Could it be that the Ets-1 protein is not immunoprecipitated or there is very little protein in your samples? Have you tested this by re-probing the blot with anti-Ets-1? I pulled down 900 micrograms of total protein. I don't want to reprobe the blot with Ets-1 because it is the same size as the IgG heavy chain. - Could you also clarify the incubation time and temperature for the anti-phosphothreonine antibody? Blot was probed overnight in 4 degrees C. We recommend mouse brain extract as positive control for ab9340. for the experiment I am performing this would not be an appropriate positive control, showing the antibody works does not get around the fact that the heavy and light chains are showing up. I apologise for the delay and hope to hear from you soon, Thank you for responding to my submission. Again I would like to clarify what the problem was. I loaded as a control a lane with only Ets-1 Antibody. This should have been blank except that there are the heavy and light chains in this lane. This indicates that the Ets-1 antibody is being bound by your phosphothreonine antibody. I ordered a biotinylated antibody to circumvent the issue of the heavy chain interference. Either your antibody is not correctly biotinlyated or for whatever reason it is binding the Ets-1 antibody. In either case, it is not performing as indicated on the product sheet and I would like either a replacement tube showing the quality control that it has been properly biotinylated or a suggestion from you that will resolve this issue. Thank you,

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    Answer

    Thank you very much for clarifying your protocol, I am sorry for the delay and would suggest to try to change the blocking step: try 5% milk 1hr or no blocking , it is possible that the antibody is binding non specifically due to the BSA. I would also recommend checking the presence of the Ets-1 protein in your samples by a simple western blotting. If you still have problems please let me know and I will send you a replacement vial,

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    Question

    BATCH NUMBER -- NOT SPECIFIED -- ORDER NUMBER P640303 DESCRIPTION OF THE PROBLEM Used Ets-1 antibody for immunoprecipitation and then used your antibody against phospho-threonine (biotinylated) on the western blot. As a control I loaded only the Ets-1 antibody. The "secondary" used was streptavidin HRP and the signal was detected with ECL. In all the lanes there was only the bands corresponding to the heavy and light chain of the antibody. The control Ets-1 antibody lane had the heavy and light chain as well. I assumed that the point of using a biotinylated antibody was to prevent picking up the heavy and light chain, since the protein I am interested in, Ets-1 is 54kd, and masked behind the heavy chain. I have performed this experiment two times, the second time loading the control Ets-1 antibody lane, and each time I get the same results. SAMPLE Cell lysate from U251 glioma cells treated with HGF at 30 minutes, 60 minutes, 3 hours and 6 hours. PRIMARY ANTIBODY Anti-phosphothreonine ab9340 Abcam product. 1:500 dilution in 10 mls of 5% BSA-PBS-Tween. The first experiment I did 1:50 dilution and same results SECONDARY ANTIBODY streptavidin HRP, no secondary antibody DETECTION METHOD ECL from amersham POSITIVE AND NEGATIVE CONTROLS USED negative control=lane from immunoprecipitation that did not receive antibody; this lane is empty. second negative control=lane containing only Ets-1 antibody same amount used for pull down; this lane contains heavy and light chain from IgG. postive control=lysate from glioma cells; this lane contains a ladder of bands indicating the antibody recognizes phosphothreonines in the lysate. ANTIBODY STORAGE CONDITIONS kept at 4 degrees C since arrival in our lab SAMPLE PREPARATION Lysed in RIPA with protease inhibitors, sheared with needle, pulled down with Ets-1 antibody, boiled 5 minutes loaded on tris gly gel AMOUNT OF PROTEIN LOADED 450 ug of protein ELECTROPHORESIS/GEL CONDITIONS 8-16% tris gly reducing, run two hours at 125 volts TRANSFER AND BLOCKING CONDITIONS Transferred to membrane for 2 hours at 25 volts in tris-gly transfer buffer with 10% methanol. blocked in 5%BSA in PBS-Tween(0.1%) for one hour HOW MANY TIMES HAVE YOU TRIED THE APPLICATION? 2 HAVE YOU RUN A "NO PRIMARY" CONTROL? No DO YOU OBTAIN THE SAME RESULTS EVERY TIME? Yes WHAT STEPS HAVE YOU ALTERED? second time added the additional control of antibody only, and diluted the abcam antibody ten fold ADDITIONAL NOTES I am quite disappointed by these results, I spent five days (one of which was 12 hours long) on the experiment, used quite a bit of money due to multiple treatments with growth factor, not to mention pull down antibody, beads and phospho specific antibody. This experiment is very important in a string of experiments and I would really like to resolve this antibody problem so I can obtain the results of this experiment. Thank you and I look forward to any comments you have.

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    Answer

    I'm very sorry to hear you are having a problem with ab9340. Thank you for taking the time to fill in the questionnaire, could you please clarify: -which antibody was used for the immunoprecipitation step(we have on anti-Ets-1 antibody but it has not been tested in IP). -Has the antibody purchased been tested in IP? - Could it be that the Ets-1 protein is not immunoprecipitated or there is very little protein in your samples? Have you tested this by re-probing the blot with anti-Ets-1? - Could you also clarify the incubation time and temperature for the anti-phosphothreonine antibody? We recommend mouse brain extract as positive control for ab9340. I apologise for the delay and hope to hear from you soon,

    Read More

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