Overview

  • Product name
    Anti-Phosphothreonine antibody
    See all Phosphothreonine primary antibodies
  • Description
    Rabbit polyclonal to Phosphothreonine
  • Host species
    Rabbit
  • Specificity
    Recognize proteins phosphorylated on threonine residues. Not cross-reacted with phosphotyrosine.
  • Tested applications
    Suitable for: WB, IP, ELISA, IHC-Frmore details
  • Species reactivity
    Reacts with: Species independent
  • Immunogen

    Chemical/ Small Molecule conjugated to KLH.

  • Positive control
    • Use mouse spleen lysate treated with sodium vanadate or mouse brain lysate for WB. Synthetic phosphopeptide (phosphorylated on threonine) for ELISA.

Properties

Applications

Our Abpromise guarantee covers the use of ab9337 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 Use a concentration of 2 µg/ml.
IP Use a concentration of 10 µg/ml. Use at a concentration of 10 µg/mg. Acetone precipitation of the protein extract followed by SDS denaturation is recommended for successful immunoprecipitation.
ELISA Use a concentration of 0.5 µg/ml.
IHC-Fr Use at an assay dependent concentration.

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

  • Immunoblotting of fetal mouse brain extract (125 ug - A and 25 ug - B)

    Immunoblotting of fetal mouse brain extract (125 ug - A and 25 ug - B)
  • Antibody Capture ELISA
    Label: immobilized antigen

References

This product has been referenced in:
  • Davison G  et al. Zinc carnosine works with bovine colostrum in truncating heavy exercise-induced increase in gut permeability in healthy volunteers. Am J Clin Nutr 104:526-36 (2016). Read more (PubMed: 27357095) »
  • Yang JH  et al. Differential regulation of the histone chaperone HIRA during muscle cell differentiation by a phosphorylation switch. Exp Mol Med 48:e252 (2016). Read more (PubMed: 27515126) »
See all 17 Publications for this product

Customer reviews and Q&As

1-9 of 9 Abreviews or Q&A

Application
Western blot
Loading amount
50 µg
Gel Running Conditions
Non-reduced Denaturing (10)
Sample
Human Cell lysate - whole cell (Immunoprecipitad protein phosphorylated at Threoni)
Specification
Immunoprecipitad protein phosphorylated at Threoni
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C

Dr. Eleni Petsalaki

Verified customer

Submitted Feb 27 2014

Answer



1) The antibody has been tested for phosphoserine in a competitive ELISA. The cross-reactivity is about 10% to phosphoserine, 25% to phosphotyrosine.



2) The relative affinity was not tested.



3) 125 ng of phosphorylated BSA is required for the detection with WB method.

Read More

Answer

Thank you for contacting us.

The ab9337 recognizes proteins phosphorylated on threonine residues. It does not cross-react with phosphotyrosine.

For ab15556, this antibody recognizes serine, threonine, and tyrosine phosphorylated proteins. It may cross-react with other phosphorylated amino acids.

For ab17464, it detects phosphoserine or phosphothreonine in the context of tyrosine, tryptophan or phenylalanine at the -1 position or phenylalanine at the +1 position. The antibody does not recognize the nonphosphorylated form of these motifs, nor does it recognize other phosphoserine or phosphothreonine containing proteins and peptides.

So I probably wouldn't recommend ab17464 for your purposes, and it seems that ab15556 may be the best fit.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Mouse Purified protein (Hepa1-6 hepatoma cells)
Loading amount
1000 µg
Specification
Hepa1-6 hepatoma cells
Treatment
1 nM insulin for 15 min/no insulin
Gel Running Conditions
Reduced Denaturing
Blocking step
BSA as blocking agent for 30 minute(s) · Concentration: 3% · Temperature: 25°C

Abcam user community

Verified customer

Submitted May 12 2012

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (A431)
Loading amount
1e+006 cells
Specification
A431
Treatment
calyculin A 1 h
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5%

Abcam user community

Verified customer

Submitted Dec 15 2006

Answer

You may want to try a nitrocellulose membrane instead - nitrocellulose membranes are considered to give less background compared to PVDF membranes. Please let me know if you have any additional questions.

Read More

Answer

Thank you for your patience. I have enquired with the originator of this antibody to see if there have been any other complaints regarding the lot that you received and I'm still waiting to hear back. Did you try decreasing the amount of primary that you are using? If not, try it at 2 ug/ml and incubate for one hour at RT rather than overnight. I know that you said that you don't believe that it is the secondary, but try incubating with the secondary only (no primary) - you should not see any background at all. Also, I'm not sure if you mentioned this already, but are you using a nitrocellulose membrane? Anyway, let me know how the above suggestions work out for you and then we can proceed from there.

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Question

Thank you for contacting me and helping me on this. Here are the answers to your questions. 1. Please describe the problem (high background, wrong band size, > more band> s, no band etc). The background is pretty high. When I expose the film for 2 min, it's totally black and I could see the membrane was green in the dark room. When I exposed for 10 sec, I could see some bands out of the dark background, but not the one I expected to see. And seems they were similar although my samples were treated with different hormones. > 2. On what material are you testing the antibody in WB? > â?¢Species? rat > â?¢Cell extract/ Nuclear extract? cell lysate > â?¢Purified protein? Please refer to the attached film, the left panel is protein after immunoprecipitation, it's relatively pure (we use Ab-FLAG for IP, and we got very strong band in WB with Ab-FLAG, so there is no possibility that the proteins were lost before the WB). The right panel is straight WB without IP process. > â?¢Recombinant protein? Yes, with Flag tag > 3. How much protein did you load? > â?¢How did you prepare the lysate for the analysis (protease > inhibito> rs etc)? I used ~250ug protein in IP process, so it¡¯s hard to say in the WB, probably 150ug? In the straight WB (right panel), I used 90ug protein. It's lysed in RIPA buffer, with protease inhibitor (Sigma), phosphatase inhibitor I and phosphatase inhibitor II (Sigma). > â?¢Did you heat the samples? Heat at 100oC for 5min > > 4. Primary Antibody > â?¢Specification (in which species was it raised against)? Rabbit polyclonal to phosphothreonine (Abcam product cold: ab9337) > â?¢At what dilution(s) have you tested this antibody? 4ug/ml as recommended, in 5% BSA TBST > â?¢Incubation time, wash step? Overnight at 4oC, rinse the blot in TBST and then wash twice for 5min each and twice for 15 min at RT (as recommended in your online protocol) > 5. Secondary Antibody > â?¢Specification (in which species was it raised against)? goat anti rabbit > â?¢At what dilution(s) have you tested this antibody? 1:15,000 in 5% BSA TBST > â?¢Incubation time, wash step? 1hr at RT, wash the blot in the same way as recommended > â?¢Do you know whether the problems you are experiencing come > from th> e secondary? I don't know. But I used your Ab against phosphoserine before (Abcam cat: Ab9332), it's very dark too, while I used other primary Ab and this 2nd Ab, it's fine. > 6. What detection method are you using? SuperSignal West Pico Chemiluminescent for detection of HRP ( PIERCE) > 7. Background bands > â?¢Have you eliminated the possibility that any background > bands coul> d be due to the secondary antibody? (Run a â??No > primaryâ??con> trol) We tried this 2nd Ab for other WB in our lab, no this kind of problem. > â?¢Is the blocking step sufficient? (We recommend blocking the > membra> ne by adding 20 ml of blocking buffer (5% non-fat dry milk, 0.1% > Tween-20 i> n TBS). Incubate for 2 h at room temperature or overnight at 4°C > with > agitation) I called your company before this experiment, they mentioned the protocol online for this Ab, which actually is a general one and only ask 1h incubation of 2nd Ab at RT. https://www.abcam.com/index.html? pageconfig=view_protocol&pid=161 > â?¢Are your washing steps sufficiently stringent? (Multiple > short was> hes are more effective than fewer longer wash steps) As mentioned in the answer to Question 4 above, I washed as recommended. > â?¢At what size are the bands migrating? Could they be > degradation pr> oducts of your target? I used protease inhibitor both during sample collection and IP. I didn¡¯t see clear bands at lower weight either. > â?¢Please provide an image of your blot (as an e-mail > attachment, a f> axed image is not sufficient) Attached. Exposure is 10 sec (up) and 3 sec (down). > 8. Optimization attempts > â?¢How many times have you tried the Western? For this one, I tried this time. But I tried 2 times with Ab- PS (Abcam 9332) from your company, also dark. I also tried Ab-PS from RDI (Research diagnostics Inc.), also dirty. > â?¢Do you obtain the same results every time e.g. are > background band> s always in the same place? For this Ab, I only tried once. > â?¢What steps have you altered? > 9. Did you apply positive and negative controls along with the > samples? Ple> ase specify. Nope. However, I used samples with different hormone treatment. I did similar experiment, in stead of WB, I send the gel after SDS-PAGE for mass spectrometry and they detect phosphorylation only with some hormone treatment. So I guess you can say I have both positive and negative samples. BTW, I was told in other protocol that for antibody against phosphoS/T/T or phosphoprotein, milk is not recommended? Why not? What will be the results if I used BSA for your Ab-PT?

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Answer

Thank you very much for the details that you have provided and for your patience. I think that the problem is due to the blocking step. For phospho-specific antibodies you should use BSA as the blocking agent, and never use milk. This is because kinases in the milk will nonspecifically phosphorylate proteins in your sample. So try BSA as your block and let me know how it turns out.

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Answer

We did find the anti-phoserine and anti-phosphothreonine antibodies bind to phosvitin, a known phosphoprotein. The anti-phosphoserine/threonine also binds to phosho-serine/threonine in a peptide. I am very sorry to inform you that we do not stock the target proteins.

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