All lanes : Anti-Securin antibody [EPR3240] (ab79546) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : PTTG1 knockout HEK-293T cell lysate
Lane 3 : Daudi cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 22 kDa
Observed band size: 28 kDa why is the actual band size different from the predicted?

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 : Anti-Securin antibody [EPR3240] (ab79546) at 1/1000 dilution (Purified)

Lane 1 : Daudi (Human Burkitt's lymphoma lymphoblast) whole cell lysates
Lane 2 : HCT 116 (Human colorectal carcinoma epithelial cell) whole cell lysates

Lysates/proteins at 15 µg per lane.

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

Predicted band size: 22 kDa
Observed band size: 28 kDa why is the actual band size different from the predicted?

All lanes : Anti-Securin antibody [EPR3240] (ab79546) at 1/20000 dilution ((unpurified))

Lane 1 : Daudi (Human Burkitt's lymphoma cell line) cell lysate
Lane 2 : HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 22 kDa
Observed band size: 28 kDa why is the actual band size different from the predicted?
Additional bands at: 25 kDa. We are unsure as to the identity of these extra bands.

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.

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 Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling Securin with purified ab79546 at 1/30 dilution (10 µg/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilized with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 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).

Unpurified ab79546 at 1/100 dilution staining Securin in paraffin-embedded human testis tissue by Immunohistochemistry.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

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).

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).