Anti-Cdc25C 抗体 [E302]

• ICC/IF

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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).

• Flow Cyt (Intra)

AbReview31366****

Flow Cytometry (Intracellular) - Anti-Cdc25C antibody [E302] (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 image is courtesy of an Abreview submitted by Brandon White.

• IHC-P

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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.

• IHC-P

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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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Cdc25C antibody [E302] (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 Fluorr^® 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.

• IP

Unknown

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.

All lanes:

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

Predicted band size: 53 kDa

Observed band size: 60 kDa

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• WB

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Western blot - Anti-Cdc25C antibody [E302] (AB32444)

Blocking and diluting buffer 5% NFDM/TBST

All lanes:

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

All lanes:

HeLa (human cervix adenocarcinoma) whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 53 kDa

Observed band size: 60 kDa

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• WB

Lab

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.

All lanes:

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

Predicted band size: 53 kDa

false

• WB

Lab

Western blot - Anti-Cdc25C antibody [E302] (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.

All lanes:

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

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

CDC25C knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human CDC25C knockout HeLa cell line (<a href='/products/cell-lines/human-cdc25c-knockout-hela-cell-line-ab265189'>ab265189</a>)

Predicted band size: 53 kDa

Observed band size: 58 kDa

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• WB

Unknown

Western blot - Anti-Cdc25C antibody [E302] (AB32444)

All lanes:

Western blot - Anti-Cdc25C antibody [E302] (ab32444) at 1/5000 dilution

All lanes:

HeLa cell lysate

Predicted band size: 53 kDa

Observed band size: 60 kDa

false

• WB

Unknown

Western blot - Anti-Cdc25C antibody [E302] (AB32444)

Blocking and diluting buffer 5% NFDM/TBST

All lanes:

Western blot - Anti-Cdc25C antibody [E302] (ab32444) at 1/5000 dilution

Lane 1:

K562 (human chronic myelogenous leukemia) whole cell lysate at 20 µg

Lane 2:

HEK293 (human embryonic kidney) whole cell lysates at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 53 kDa

Observed band size: 60 kDa

false