Recombinant Anti-Cdc25C antibody [E302] - BSA and Azide free (ab232553)

This data was developed using the same antibody clone in a different buffer formulation (ab32444).

  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.

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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). 

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Blocking buffer and concentration: 5% NFDM/TBST.

Diluting buffer and concentration: 5% NFDM /TBST.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32444).

Intracellular 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. 

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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: Human 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 1/2,500 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 1/10,000 dilution for 1 hour at room temperature before imaging.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32444).

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32444).

Intracellular 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. 

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32444). 

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