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    cdc25c-antibody-e302-ab32444.pdf

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Cell Biology Cell Cycle Kinases/Phosphatases Phosphatases
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Validated using a knockout cell lineRecombinantRabMAb

Recombinant Anti-Cdc25C antibody [E302] (ab32444)

  • Datasheet
  • SDS
Reviews (3) Submit a question References (18)

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Western blot - Anti-Cdc25C antibody [E302] (ab32444)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cdc25C antibody [E302] (ab32444)
  • Immunocytochemistry/ Immunofluorescence - Anti-Cdc25C antibody [E302] (ab32444)
  • Immunoprecipitation - Anti-Cdc25C antibody [E302] (ab32444)
  • Western blot - Anti-Cdc25C antibody [E302] (ab32444)
  • Flow Cytometry - Anti-Cdc25C antibody [E302] (ab32444)
  • Western blot - Anti-Cdc25C antibody [E302] (ab32444)
  • Western blot - Anti-Cdc25C antibody [E302] (ab32444)
  • Western blot - Anti-Cdc25C antibody [E302] (ab32444)
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cdc25C antibody [E302] (ab32444)
  • Flow Cytometry - Anti-Cdc25C antibody [E302] (ab32444)
  • Anti-Cdc25C antibody [E302] (ab32444)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rabbit monoclonal [E302] to Cdc25C
  • Suitable for: WB, IHC-P, ICC/IF, IP, Flow Cyt
  • Knockout validated
  • Reacts with: Human

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Overview

  • Product name

    Anti-Cdc25C antibody [E302]
    See all Cdc25C primary antibodies
  • Description

    Rabbit monoclonal [E302] to Cdc25C
  • Host species

    Rabbit
  • Specificity

    The antibody can also detect splice isoform 2, 4 and 5 of human Cdc25C, based on sequence homology.
  • Tested Applications & Species

    Application Species
    Flow Cyt
    Human
    ICC/IF
    Human
    IHC-P
    Human
    IP
    Human
    WB
    Human
    See all applications and species data
  • Immunogen

    Synthetic peptide within Human Cdc25C aa 1-100 (N terminal). The exact sequence is proprietary.

  • Positive control

    • WB: HeLa, Hap1, K562 and HEK293 cell lysates. IHC-P: Human pancreas and urinary bladder carcinoma tissue. ICC/IF: HeLa cells. IP: HeLa cells. Flow Cyt: HeLa cells.
  • General notes

    Mouse, Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.

     

    This product is a recombinant monoclonal antibody, which offers several advantages including:

    • - High batch-to-batch consistency and reproducibility
    • - Improved sensitivity and specificity
    • - Long-term security of supply
    • - Animal-free production
    For more information see here.

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

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing the problem with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation.

    One factor contributing to the crisis is the use of antibodies that are not suitable. This can lead to misleading results and the use of incorrect data informing project assumptions and direction. To help address this challenge, we have introduced an application and species grid on our primary antibody datasheets to make it easy to simplify identification of the right antibody for your needs.

    Learn more here.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle.
  • Storage buffer

    pH: 7.20
    Preservative: 0.01% Sodium azide
    Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    E302
  • Isotype

    IgG
  • Research areas

    • Cell Biology
    • Cell Cycle
    • Kinases/Phosphatases
    • Phosphatases
    • Epigenetics and Nuclear Signaling
    • Cell cycle
    • Kinases/Phosphatases
    • Phospatases

Associated products

  • Alternative Versions

    • Alexa Fluor® 488 Anti-Cdc25C antibody [E302] (ab205425)
    • Alexa Fluor® 647 Anti-Cdc25C antibody [E302] (ab205426)
    • HRP Anti-Cdc25C antibody [E302] (ab205734)
    • Anti-Cdc25C antibody [E302] - BSA and Azide free (ab232553)
  • Compatible Secondaries

    • Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077)
    • Goat Anti-Rabbit IgG H&L (HRP) (ab205718)
    • Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773)
  • Conjugation kits

    • PE / R-Phycoerythrin Conjugation Kit - Lightning-Link® (ab102918)
    • APC Conjugation Kit - Lightning-Link® (ab201807)
  • Isotype control

    • Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730)
  • KO cell lines

    • Human CDC25C knockout HeLa cell line (ab265189)
  • KO cell lysates

    • Human CDC25C knockout HeLa cell lysate (ab257387)
  • Positive Controls

    • HeLa whole cell lysate (ab29545)
  • Recombinant Protein

    • Recombinant Human Cdc25C protein (ab158076)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab32444 in the following tested applications.

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

Guaranteed

Tested applications are guaranteed to work and covered by our Abpromise guarantee.

Predicted

Predicted to work for this combination of applications and species but not guaranteed.

Incompatible

Does not work for this combination of applications and species.

Application Species
Flow Cyt
Human
ICC/IF
Human
IHC-P
Human
IP
Human
WB
Human
Application Abreviews Notes
WB (2)
1/1000 - 1/5000. Detects a band of approximately 60 kDa (predicted molecular weight: 53 kDa).
IHC-P
1/2500. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

For unpurified, use 1/250 - 1/500.

ICC/IF
1/250 - 1/500.
IP
1/30 - 1/80.
Flow Cyt (1)
Use at an assay dependent concentration.
Notes
WB
1/1000 - 1/5000. Detects a band of approximately 60 kDa (predicted molecular weight: 53 kDa).
IHC-P
1/2500. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

For unpurified, use 1/250 - 1/500.

ICC/IF
1/250 - 1/500.
IP
1/30 - 1/80.
Flow Cyt
Use at an assay dependent concentration.

Target

  • Function

    Functions as a dosage-dependent inducer in mitotic control. It is a tyrosine protein phosphatase required for progression of the cell cycle. It directly dephosphorylates CDK1 and activate its kinase activity.
  • Sequence similarities

    Belongs to the MPI phosphatase family.
    Contains 1 rhodanese domain.
  • Developmental stage

    Expressed predominantly in G2 phase.
  • Post-translational
    modifications

    Phosphorylated by CHK1 on Ser-216. This phosphorylation creates a binding site for 14-3-3 protein and inhibits the phosphatase. Phosphorylated by PLK4.
  • Cellular localization

    Nucleus.
  • Target information above from: UniProt accession P30307 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links

    • Entrez Gene: 995 Human
    • Omim: 157680 Human
    • SwissProt: P30307 Human
    • Unigene: 656 Human
    • Alternative names

      • CDC 25 antibody
      • Cdc 25C antibody
      • CDC25 antibody
      • CDC25C antibody
      • Cell division cycle 25 homolog C antibody
      • Cell division cycle 25C antibody
      • Cell division cycle 25C protein antibody
      • Dual specificity phosphatase Cdc25C antibody
      • M phase inducer phosphatase 3 antibody
      • M-phase inducer phosphatase 3 antibody
      • Mitosis inducer CDC25 antibody
      • MPIP3 antibody
      • MPIP3_HUMAN antibody
      • Phosphotyrosine phosphatase antibody
      • PPP1R60 antibody
      • protein phosphatase 1, regulatory subunit 60 antibody
      see all

    Images

    • Western blot - Anti-Cdc25C antibody [E302] (ab32444)
      Western blot - Anti-Cdc25C antibody [E302] (ab32444)
      All lanes : Anti-Cdc25C antibody [E302] (ab32444) at 1/1000 dilution

      Lane 1 : Wild-type HeLa cell lysate
      Lane 2 : CDC25C knockout HeLa cell lysate

      Lysates/proteins at 20 µg per lane.

      Performed under reducing conditions.

      Predicted band size: 53 kDa
      Observed band size: 58 kDa why is the actual band size different from the predicted?



      Lanes 1- 2: Merged signal (red and green). Green - ab32444 observed at 58 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

       ab32444 was shown to react with Cdc25C in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265189 (knockout cell lysate ab257387) was used. Wild-type HeLa and CDC25C knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab32444 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cdc25C antibody [E302] (ab32444)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cdc25C antibody [E302] (ab32444)

      Immunohistochemical analysis of paraffin embedded human pancreas tissue section labelling Cdc25C with purified ab32444 at dilution of 1/2500. The secondary antibody used was Goat Anti-Rabbit IgG H&L (HRP) (ab97051), at dilution of 1/500. The sample was counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control and is shown in the inset.

    • Immunocytochemistry/ Immunofluorescence - Anti-Cdc25C antibody [E302] (ab32444)
      Immunocytochemistry/ Immunofluorescence - Anti-Cdc25C antibody [E302] (ab32444)

      Immunocytochemistry/Immunofluorescence analysis of HeLa (human cervix adenocarcinoma) cells labelling Cdc25C with purified ab32444 at 1/400. Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. The cells were co-stained with ab7291, a mouse anti-tubulin antibody (1/1000) using ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/1000) as the secondary. Nuclei couterstained with DAPI (blue).

      For negative control 1, rabbit primary antibody was used, followed by anti-mouse secondary antibody (ab150120). For negative control 2, mouse primary antibody (ab7291) was used followed by anti-rabbit secondary antibody (ab150077). 

    • Immunoprecipitation - Anti-Cdc25C antibody [E302] (ab32444)
      Immunoprecipitation - Anti-Cdc25C antibody [E302] (ab32444)

      Ab32444 (purified) at 1/30 immunoprecipitating Cdc25C in HeLa (human cervix adenocarcinoma) whole cell lysate.

      Lane 1 (input): HeLa (human cervix adenocarcinoma) whole cell lysate

      Lane 2 (+): ab32444 + HeLa (human cervix adenocarcinoma) whole cell lysate

      Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32444 in HeLa (human cervix adenocarcinoma) whole cell lysate

      For western blotting, ab131366 VeriBlot for IP Detection Reagent (HRP) was used for detection (1/10000).

      Blocking buffer and concentration: 5% NFDM/TBST.

      Diluting buffer and concentration: 5% NFDM /TBST.

    • Western blot - Anti-Cdc25C antibody [E302] (ab32444)
      Western blot - Anti-Cdc25C antibody [E302] (ab32444)

      Lane 1: Wild-type HAP1 cell lysate (20 µg)
      Lane 2: Cdc25C knockout HAP1 cell lysate (20 µg)
      Lane 3: HeLa cell lysate (20 µg)
      Lane 4: Hu bladder cell lysate (20 µg)
      Lanes 1 - 4: Merged signal (red and green). Green - ab32444 observed at 55 kDa. Red - loading control, ab8245, observed at 37 kDa.

      ab32444 was shown to recognize Cdc25C when Cdc25C knockout samples were used, along with additional cross-reactive bands. Wild-type and Cdc25C knockout samples were subjected to SDS-PAGE. ab32444 and ab8245 (loading control to GAPDH) were diluted at 1/2500 and 1/10 000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 h at room temperature before imaging.

    • Flow Cytometry - Anti-Cdc25C antibody [E302] (ab32444)
      Flow Cytometry - Anti-Cdc25C antibody [E302] (ab32444)

      Flow Cytometry analysis of K562 (human chronic myelogenous leukemia) cells labelling Cdc25C with purified ab32444 at 1/180 (red). Cells were fixed with 4% paraformaldehyde. Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

    • Western blot - Anti-Cdc25C antibody [E302] (ab32444)
      Western blot - Anti-Cdc25C antibody [E302] (ab32444)
      All lanes : Anti-Cdc25C antibody [E302] (ab32444) at 1/5000 dilution (purified)

      Lane 1 : K562 (human chronic myelogenous leukemia) whole cell lysate
      Lane 2 : HEK293 (human embryonic kidney) whole cell lysates

      Lysates/proteins at 20 µg per lane.

      Secondary
      All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

      Predicted band size: 53 kDa
      Observed band size: 60 kDa why is the actual band size different from the predicted?



      Blocking and diluting buffer 5% NFDM/TBST

    • Western blot - Anti-Cdc25C antibody [E302] (ab32444)
      Western blot - Anti-Cdc25C antibody [E302] (ab32444)
      Anti-Cdc25C antibody [E302] (ab32444) at 1/1000 dilution (purified) + HeLa (human cervix adenocarcinoma) whole cell lysate at 20 µg

      Secondary
      Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

      Predicted band size: 53 kDa
      Observed band size: 60 kDa why is the actual band size different from the predicted?



      Blocking and diluting buffer 5% NFDM/TBST

    • Western blot - Anti-Cdc25C antibody [E302] (ab32444)
      Western blot - Anti-Cdc25C antibody [E302] (ab32444)
      Anti-Cdc25C antibody [E302] (ab32444) at 1/5000 dilution (unpurified) + HeLa cell lysate

      Predicted band size: 53 kDa
      Observed band size: 60 kDa why is the actual band size different from the predicted?

    • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cdc25C antibody [E302] (ab32444)
      Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cdc25C antibody [E302] (ab32444)

      Immunohistochemical analysis of paraffin-embedded human urinary bladder carcinoma unpurified ab32444 at 1/250 dilution.

    • Flow Cytometry - Anti-Cdc25C antibody [E302] (ab32444)
      Flow Cytometry - Anti-Cdc25C antibody [E302] (ab32444)This image is courtesy of an Abreview submitted by Dr Brandon White

      Flow cytometry analysis of HeLa cells, staining Cdc25C with unpurified ab32444.

      Cells were fixed with formaldehyde and permeabilized with 90% methanol. Samples were incubated with primary antibody (1/20 in PBS + 10% goat serum) for 1 hour at 23°C. A FITC-conjugated goat anti-rabbit polyclonal IgG (1/1000) was used as the secondary antibody.

      See Abreview

    • Anti-Cdc25C antibody [E302] (ab32444)
      Anti-Cdc25C antibody [E302] (ab32444)

    Protocols

    To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

    Click here to view the general protocols

    Datasheets and documents

    • SDS download

    • Datasheet download

      Download

    References (18)

    Publishing research using ab32444? Please let us know so that we can cite the reference in this datasheet.

    ab32444 has been referenced in 18 publications.

    • Cao Y  et al. Mining the Prognostic Value of HNRNPAB and Its Function in Breast Carcinoma. Int J Genomics 2020:3750673 (2020). PubMed: 32509843
    • Pan Z  et al. Cinobufagin Induces Cell Cycle Arrest at the G2/M Phase and Promotes Apoptosis in Malignant Melanoma Cells. Front Oncol 9:853 (2019). PubMed: 31552178
    • Wu M  et al. A PLK1 kinase inhibitor enhances the chemosensitivity of cisplatin by inducing pyroptosis in oesophageal squamous cell carcinoma. EBioMedicine 41:244-255 (2019). PubMed: 30876762
    • Ying H  et al. Overexpression of Zwint predicts poor prognosis and promotes the proliferation of hepatocellular carcinoma by regulating cell-cycle-related proteins. Onco Targets Ther 11:689-702 (2018). PubMed: 29440916
    • Shrestha D  et al. Actin Dysfunction Induces Cell Cycle Delay at G2/M with Sustained ERK and RSK Activation in IMR-90 Normal Human Fibroblasts. Mol Cells 41:436-443 (2018). PubMed: 29754473
    View all Publications for this product

    Customer reviews and Q&As

    Show All Reviews Q&A
    Submit a review Submit a question

    1-3 of 3 Abreviews or Q&A

    Western blot abreview for Anti-Cdc25C antibody [E302]

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (HeLa cell lysate)
    Loading amount
    20 µg
    Specification
    HeLa cell lysate
    Gel Running Conditions
    Reduced Denaturing (4-12% Bis-Tris)
    Blocking step
    Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
    Read More

    Abcam user community

    Verified customer

    Submitted May 15 2013

    Western blot abreview for Anti-Cdc25C antibody [E302]

    Average
    Abreviews
    Abreviews
    abreview image
    Application
    Western blot
    Sample
    Human Cell lysate - whole cell (Human Fibroblast)
    Loading amount
    15 µg
    Specification
    Human Fibroblast
    Gel Running Conditions
    Reduced Denaturing (4-12% Bis-Tris Nupage gel)
    Blocking step
    BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
    Read More

    Abcam user community

    Verified customer

    Submitted Feb 25 2013

    Flow Cytometry abreview for Anti-Cdc25C antibody [E302]

    Excellent
    Abreviews
    Abreviews
    abreview image
    Application
    Flow Cytometry
    Sample
    Human Cell (HeLa Cells)
    Specification
    HeLa Cells
    Preparation
    Cell harvesting/tissue preparation method: Trypsin/EDTA
    Sample buffer: Complete Media then PBS
    Fixation
    Formaldehyde
    Permeabilization
    Yes - 90% Methanol
    Read More

    Dr. Brandon White

    Verified customer

    Submitted Aug 30 2012

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