Anti-Phosphoserine/threonine/tyrosine antibody [SPM101] (ab15556)

Overview

  • Product name
    Anti-Phosphoserine/threonine/tyrosine antibody [SPM101]
  • Description
    Mouse monoclonal [SPM101] to Phosphoserine/threonine/tyrosine
  • Host species
    Mouse
  • Tested applications
    Suitable for: IHC - Wholemount, Functional Studies, ICC/IF, Flow Cyt, ELISA, WB, IP, IHC-P, IHC-Frmore details
  • Species reactivity
    Reacts with: Species independent
  • Immunogen

    Phosphoserine, phosphothreonine, and phosphotyrosine conjugated to protein carriers.

  • Positive control
    • Breast carcinoma

Properties

Applications

Our Abpromise guarantee covers the use of ab15556 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
IHC - Wholemount 1/200.
Functional Studies Use at an assay dependent concentration.
ICC/IF Use at an assay dependent concentration.
Flow Cyt Use 1µg for 106 cells.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

ELISA Use at an assay dependent concentration.
WB 1/50.

Milk derived blocking solutions reportedly contain phosphoproteins that may inhibit phosphoamino acid antibody binding and therefore should be avoided.

IP Use at 2 µg/mg of lysate.
IHC-P 1/20. Boil tissue sections in 10mM citrate, pH 6.0 for 10 min followed by cooling at RT for 20 min.
IHC-Fr Use at an assay dependent concentration. PubMed: 20421451

Target

  • Relevance
    Protein phosphorylation provides a signalling system that can be thought of as a kind of protein on/off switch for many cellular signalling pathways. Phosphorylation is observed on serine, threonine, tyrosine and histidine residues. Cellular networks underlying phosphorylation can be very complex and often occurs on multiple distinct sites on a given protein. Phospho-specific antibodies are becoming critical reagents both for basic research and for clinical diagnosis.

Images

  • Human breast carcinoma stained with ab15556. Immunohistochemistry (FFPE-sections).
  • Overlay histogram showing MCF7 cells stained with ab15556 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab15556, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in MCF7 cells fixed with 80% methanol (5 min)/permeabilized in 0.1% PBS-Tween used under the same conditions.

References

This product has been referenced in:
  • Pereira C  et al. Sit4p-mediated dephosphorylation of Atp2p regulates ATP synthase activity and mitochondrial function. Biochim Biophys Acta 1859:591-601 (2018). Read more (PubMed: 29719209) »
  • Zhu G  et al. Sublytic C5b-9 Induces Glomerular Mesangial Cell Apoptosis through the Cascade Pathway of MEKK2-p38 MAPK-IRF-1-TRADD-Caspase 8 in Rat Thy-1 Nephritis. J Immunol 198:1104-1118 (2017). Read more (PubMed: 28039298) »
See all 16 Publications for this product

Customer reviews and Q&As

1-10 of 25 Abreviews or Q&A

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (HEK293)
Permeabilization
Yes - 0.1% Triton X-100, 10 min RT
Specification
HEK293
Blocking step
Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Jun 18 2018

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (colon cell)
Permeabilization
Yes - 1% triton x100
Specification
colon cell
Blocking step
BSA as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 37°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Sep 05 2016

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - nuclear (293T)
Gel Running Conditions
Non-reduced Non-Denaturing (Native)
Loading amount
80 µg
Treatment
damage stress
Specification
293T
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 37°C

Abcam user community

Verified customer

Submitted Sep 05 2016

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Mouse Tissue sections (mice pancreas tumors)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Tris EDTA pH 9
Permeabilization
Yes - Tween-20
Specification
mice pancreas tumors
Blocking step
BSA as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Aug 18 2015

Application
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample
Human Tissue sections (Human fibromatosis)
Antigen retrieval step
Heat mediated - Buffer/Enzyme Used: Tris EDTA pH 9
Permeabilization
Yes - Tween-20
Specification
Human fibromatosis
Blocking step
BSA as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Aug 18 2015

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.

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Answer

It (ab15556) is specific for phosphate groups on any serine, threonine, or tyrosine, on any protein, so it may not be specific enough for your purposes, if your samples contain other phosphorylated proteins besides EGFR.

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Question
Answer

Thank you for contacting us.

I have been in touch with the originator of this product to discuss exactly how tab15556 Anti-Phosphoserine/threonine/tyrosine antibody [SPM101] is made. Apparently the immunogen was a carrier protein (used to elicit response) which had Phosphoserine, phosphothreonine and phosphotyrosine attached. The clone was then chosen which had response to each.

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



Use our products? Submit an Abreview. Earn rewards!

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Answer




I am very pleased to hear you would like to accept our offer and test ab15556 in plants in WB. This code will give you: 1 free primary antibody before the expiration date. To redeem this offer, please submit an Abreview forgrape extractand include this code in the “Additional Comments” section so we know the Abreview is for this promotion. Please remember that submission of the Abreview is sufficient for the discount code to become active.
For more information on how to submit an Abreview, please visit the site: www.abcam.com/Abreviews.


Remember, we publish both positive and negative Abreviews on our datasheets so please submit the results of your tests. The code will be active once the Abreview has been submitted and can be redeemed in one of the following ways: 1) Call to place your order and mention the code to our customer service department; 2) Include the code in your fax order; 3) Place your order on the web and enter the promotional code.

Any feedback that you can provide will be greatly appreciated, whether positive or negative. If you have any further questions, please do not hesitate to contact us. We look forward to receiving your Abreview and wish you luck with your research.

The terms and conditions applicable to this offer can be found here: www.abcam.com/collaborationdiscount.

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Answer

Thank you for your reply.

Unfortunately, 2D gel analysis is not an application we can issue the Abreview discount for. The reason for this is that this is not an application you can review via our system.

However, I would gladly issue you the code if you would perform a WB or the like.

I hope this information is helpful to you. Please let me know this is acceptable for you.

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1-10 of 25 Abreviews or Q&A

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