Anti-Securin antibody [EPR3240]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Securin antibody [EPR3240] (AB79546)

Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Securin with purified ab79546 at 1/30 dilution (10μg/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilized with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluorr^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Securin antibody [EPR3240] (AB79546)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carcinoma tissue sections labeling Securin with purified ab79546 at 1/200 dilution (1.19 μg/ml). Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody.

Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-Securin antibody [EPR3240] (AB79546)

Intracellular flow cytometric analysis of permeabilized Jurkat (Human T cell leukemia cell line from peripheral blood) cells using unpurified ab79546 (red) or a rabbit IgG (negative) (green).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Securin antibody [EPR3240] (AB79546)

Immunofluorescent analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling securin with unpurified ab79546 at 1/250 dilution. The cells were permeabilised with 0.1% Triton X-100. Anti-rabbit Alexa Fluor^® 488 (ab150077) at 1/500 dilution was used as the secondary antibody (green). The nuclear counter stain is DAPI (blue).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-Securin antibody [EPR3240] (AB79546)

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Securin with purified ab79546 at 1/50 dilution (4.74 μg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) 1/200 (2.5 μg/ml). Goat anti rabbit IgG (Alexa Fluor® 488, ab150077) was used as the secondary antibody at 1/1000 (2 μg/ml) dilution. DAPI (blue) was used as nuclear counterstain.

PBS instead of the primary antibody was used as the secondary antibody only control.

• WB

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Western blot - Anti-Securin antibody [EPR3240] (AB79546)

Lanes 1-3 : Merged signal (red and green). Green - ab79546 observed at 28 kDa. Red - loading control, ab8245 observed at 37 kDa.

ab79546 Anti-Securin antibody [EPR3240] was shown to specifically react with Securin in wild-type HEK293T cells. Loss of signal was observed when knockout cell line ab266231 (knockout cell lysate ab257289) was used. Wild-type and Securin knockout samples were subjected to SDS-PAGE. ab79546 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 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 10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Securin antibody [EPR3240] (ab79546) at 1/1000 dilution

Lane 1:

Wild-type HEK293T cell lysate at 20 µg

Lane 2:

PTTG1 knockout HEK293T cell lysate at 20 µg

Lane 3:

Daudi cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/10000 dilution

Predicted band size: 22 kDa

Observed band size: 28 kDa

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• WB

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Western blot - Anti-Securin antibody [EPR3240] (AB79546)

All lanes:

Western blot - Anti-Securin antibody [EPR3240] (ab79546) at 1/20000 dilution

Lane 1:

Daudi (Human Burkitt's lymphoma cell line) cell lysate at 10 µg

Lane 2:

HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 22 kDa

Observed band size: 25 kDa,28 kDa

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• WB

Lab

Western blot - Anti-Securin antibody [EPR3240] (AB79546)

All lanes:

Western blot - Anti-Securin antibody [EPR3240] (ab79546) at 1/1000 dilution

Lane 1:

Daudi (Human Burkitt's lymphoma lymphoblast) whole cell lysates at 15 µg

Lane 2:

HCT 116 (Human colorectal carcinoma epithelial cell) whole cell lysates at 15 µg

Secondary

All lanes:

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

Predicted band size: 22 kDa

Observed band size: 28 kDa

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• WB

CiteAb

Western blot - Anti-Securin antibody [EPR3240] (AB79546)

Western Blotting using Anti-Securin antibody [EPR3240], ab79546. Publication image from Chen, J. et al., 2016, Nat Commun, 26743940. Legend direct from paper.

Cep57 contributes to activation of the spindle assembly checkpoint.(a,b) HeLa cells with 100 nM nocodazole treatment after co-transfection with H2B-RFP and negative control (NC)-, Cep57-, Mad1- or Mad2-siRNA for 48 h. (a) Time-lapse images of HeLa cells. The numbers indicate the time (minutes) after entry into mitosis. Arrowhead, multiple nuclei. Scale bar, 5 µm. (b) Quantification of mitotic duration of HeLa cells from a. Scatter plots show data from three independent experiments. (c) Quantification of mitotic duration of live HeLa cells with 80 nM taxol treatment after co-transfection with H2B-RFP and negative control (NC)-, Cep57-, Mad1- or Mad2-siRNA for 48 h. Scatter plots show data from three independent experiments. (d) Diagram of experimental design. The siRNA-transfected HeLa cells were synchronized by double-thymidine block, and released into nocodazole. (e–g) HeLa cells were treated as in d. (e) After 12 h nocodazole treatment, mitotic cell lysates were used to perform immunoprecipitation (IP) and western blotting assays with the indicated antibodies. WCL, whole-cell lysate. (f) Western blots of the indicated proteins in HeLa cells after the indicated nocodazole (Noc.) treatment time. (g) Quantification of the protein levels of securin and cyclin B1 from (f). The experiment was repeated three times. For b,c and g, data are mean±s.e.m. ****P<0.0001; ***P<0.001; **P<0.01; *P<0.05; NS, not significant (unpaired two-tailed Student's t-test). DIC, differential interference contrast.

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