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A synthetic peptide corresponding to residues near N-terminus of human Cdc25C.
Mouse, Rat: We have preliminary internal testing data to indicate this antibody may not react with these species. Please contact us for more information.
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.
This product is a recombinant rabbit monoclonal antibody.
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.
|WB||1/1000 - 1/5000. Detects a band of approximately 60 kDa (predicted molecular weight: 53 kDa).|
For unpurified, use 1/250 - 1/500.
|ICC/IF||1/250 - 1/500.|
ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
|IP||1/30 - 1/80.|
Blocking and diluting buffer 5% NFDM/TBST
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 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).
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.
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 (HRP) was used as the secondary antibody (1/10000).
Blocking buffer and concentration: 5% NFDM/TBST.
Diluting buffer and concentration: 5% NFDM /TBST.
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.
Blocking and diluting buffer 5% NFDM/TBST
Immunohistochemical analysis of paraffin-embedded human urinary bladder carcinoma unpurified ab32444 at 1/250 dilution.Immunohistochemical analysis of paraffin-embedded human urinary bladder carcinoma ab32444 at 1/250 dilution.
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.