All lanes : Anti-PBK/SPK antibody [EPR21982] (ab236871) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : PBK knockout HEK-293T cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 36 kDa
Observed band size: 40 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab236871 observed at 40 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

 ab236871 was shown to react with PBK/SPK in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab266827 (knockout cell lysate ab257575) was used. Wild-type HEK-293T and PBK knockout HEK-293T cell lysates were subjected to SDS-PAGE. ab236871 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) 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 testis tissue labeling PBK/SPK with ab236871 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Positive staining in germ cells of human testis (PMID:16982762; PMID:25909225) is observed. Counterstained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cell line labeling PBK/SPK with ab236871 at 1/50 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.

All lanes : Anti-PBK/SPK antibody [EPR21982] (ab236871) at 1/1000 dilution

Lane 1 : Wild-type HAP1 cell lysate
Lane 2 : PBK/SPK knockout HAP1 cell lysate
Lane 3 : HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 4 : HepG2 (human liver hepatocellular carcinoma cell line) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 36 kDa
Observed band size: 38 kDa why is the actual band size different from the predicted?


Exposure time: 15 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

The molecular weight observed is consistent with what has been described in the literature (PMID:23547718; PMID:25909225).

ab236871 was shown to specifically react with PBK/SPK in wild-type HAP1 cells as signal was lost in PBK/SPK knockout cells. Wild-type and PBK/SPK knockout samples were subjected to SDS-PAGE. ab236871 and ab181602 (Rabbit anti-GAPDH loading control) were incubated 1 hour at room temperature at 1/1000 dilution and 1/200,000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) secondary antibody at 1/100,000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-PBK/SPK antibody [EPR21982] (ab236871) at 1/1000 dilution

Lane 1 : SW480 (human colorectal adenocarcinoma cell line) whole cell lysate at 20 µg
Lane 2 : A431 (human epidermoid carcinoma cell line) whole cell lysate at 20 µg
Lane 3 : U-87 MG (human glioblastoma-astrocytoma epithelial cell line) whole cell lysate at 20 µg
Lane 4 : Rat testis lysate at 20 µg
Lane 5 : C6 (rat glial tumor cell line) whole cell lysate at 10 µg
Lane 6 : PC-12 (rat adrenal gland pheochromocytoma cell line) whole cell lysate at 10 µg

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 36 kDa
Observed band size: 38 kDa why is the actual band size different from the predicted?

Exposure time : Lanes 1-3: 26 seconds; Lane 4: 8 seconds; Lanes 5-6: 70 seconds.

Blocking/Dilution buffer: 5% NFDM/TBST.

The molecular weight observed is consistent with what has been described in the literature (PMID:23547718; PMID:25909225).

PBK/SPK was immunoprecipitated from 0.35 mg of HeLa (human epithelial cell line from cervix adenocarcinoma) whole cell lysate with ab236871 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab184276 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution.

Lane 1: HeLa whole cell lysate 10 µg (Input). 

Lane 2: ab231871 IP in HeLa whole cell lysate. 

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab231871 in HeLa whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 30 seconds.

Immunohistochemical analysis of paraffin-embedded human esophagus cancer tissue labeling PBK/SPK with ab236871 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Positive staining in human esophagus cancer (PMID: 27919968) is observed. Counterstained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunohistochemical analysis of paraffin-embedded human gastric cancer tissue labeling PBK/SPK with ab236871 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Positive staining in human gastric cancer (PMID:26894977; PMID:27898655) is observed. Counterstained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized HepG2 (human liver hepatocellular carcinoma cell line) cell line labeling PBK/SPK with ab236871 at 1/50 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/2000 dilution was used as the secondary antibody.