Overview

  • Product name
    Anti-Phosphoserine antibody [PSR-45]
    See all Phosphoserine primary antibodies
  • Description
    Mouse monoclonal [PSR-45] to Phosphoserine
  • Host species
    Mouse
  • Specificity
    The clone number has been updated from (3C171) to (PSR-45) both clone numbers name the same antibody clone.
  • Tested applications
    Suitable for: ELISA, WB, ICC/IFmore details
  • Species reactivity
    Reacts with: Species independent
  • Immunogen

    Chemical/ Small Molecule corresponding to Phosphoserine conjugated to Keyhole Limpet Haemocyanin (KLH).

Properties

Applications

Our Abpromise guarantee covers the use of ab17465 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
ELISA 1/8000 - 1/16000.
WB 1/500 - 1/1000.

(colorimetric). Do not use milk as a blocking agent or in diluents, as milk casein is phosphorylated at several serine residues. BSA is recommended instead.

ICC/IF Use a concentration of 1 µg/ml.

Target

  • Relevance
    Changes in the serine/threonine phosphorylation state of a protein in response to various external stimuli can have profound effects on cellular signal transduction, apoptosis and carcinogenesis. The reagents, including phosphorylated protein/peptides, antibodies against the phosphospecific amino acid, are important tools to explore the activation of serine, threonine or tyrosine containing proteins. An aberrant protein phosphorylation is a hallmark of human disease, and the enzymes, particularly protein kinases, which control protein phosphorylation are recognized as a major new drug target family.
  • Alternative names
    • phospho-Ser antibody
    • pS antibody
    • pSER antibody

Images

  • ICC/IF image of ab17465 stained MCF-7 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab17465, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

  • Canine brain hippocampus homogenates (10ug loaded per lane for 3 different samples) and blotted with ab17465. Western blot of canine brain hippocampus homogenates (10 ug loaded per lane for 3 different samples) separated on SDS-PAGE and blotted with ab17465.

References

This product has been referenced in:
  • Yoon YJ  et al. 2'-hydroxycinnamaldehyde inhibits cancer cell proliferation and tumor growth by targeting the pyruvate kinase M2. Cancer Lett 434:42-55 (2018). Read more (PubMed: 30009856) »
  • Li S  et al. CaMKII Potentiates Store-Operated Ca2+ Entry Through Enhancing STIM1 Aggregation and Interaction with Orai1. Cell Physiol Biochem 46:1042-1054 (2018). Read more (PubMed: 29669320) »
See all 10 Publications for this product

Customer reviews and Q&As

1-5 of 5 Abreviews or Q&A

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Cow Cell lysate - whole cell (Macrophage)
Loading amount
10 µg
Specification
Macrophage
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5%

Abcam user community

Verified customer

Submitted Dec 22 2006

Answer

Thank you for your enquiry. I was able to obtain an image. I'll send it to you in a separate email as I cannot attach it here. You will see that the antibody was tested at 1:200 in a Drosophila melanogaster whole tissue lysate (40ug/lane). There are multiple bands as there are multiple serine phosphorylated proteins. I will add the image to the online datasheet as soon as I have more confirmation on the possible identity of these bands. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

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Answer

Thank you for your enquiry. I have updated the specificity statement for ab17465 with the following information from the source of the antibody: Recognizes phosphorylated serine both as free amino acid and when coupled to carriers such as BSA or KLH. Does not crossreact with non-phosphorylated serine, phosphothreonine, phosphotyrosine, AMP or ATP. Species crossreactivity: Human, monkey, bovine, canine, rat, chicken, rat kangaroo, goose and hamster. Apparently the antigen specificity is quite high; but it is an ascites grade antibody with antibody concentration indeterminant. The affinity must not be extraordinarily high with the suggested dilution of 1:250 for Western Blotting. This is the most exact information I could find. It appears that the affinity of the antibody for its ligand was not calculated. I hope this information helps, please do not hesitate to contact us if you need any more advice or information.

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Answer

I'm sorry to hear you are having problems with ab17465. Could you please clarify what you mean with "no signal despite positive controls on western"? Did the antibody work on cell lysate in WB and stop working when your protein of interest was immunoprecipitated? Could it be that your protein of interest is not phosphorylated sufficiently to detect it by WB or total levels of the protein are very low? It is also possible that the phosphate groups are destroyed by phosphatases. Could you tell me more about the concentrations of your phosphatase inhibitors? I would recommend making sure the samples are on ice at all times and that following stimulation the cells are snap frozen to keep the protein in the phosphorylated state. If you would like to inhibit endogenous phosphatases the addition of okadaic acid should be done prior to stimulation rather than prior to lysis, but in my experience cells can suffer a lot from this chemical. The primary antibody should be used at a 1:250 dilution, as you have not detailed which dilution you have tested could you please confirm you have used this dilution? I would also recommend using TBST (tween20 0.1%) to dilute antibodies and to wash, as the water wash step may affect the antibody binding properties. Finally, I would recommend ECL+ rather than ECL as this is more sensitive, I hope these first suggestions will help you and look forward to hearing from you to help you more,

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Answer

The immunogen was phosphoserine-KLH and the antibody recognizes free phosphoserine or phosphoserine conjugated to a carrier such as KLH or BSA. There is no immunizing peptide; just a phosphorylated amino acid.

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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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