All lanes : Anti-PBK/SPK antibody [EPR21983] (ab236872) at 1/1000 dilution

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

ab236872 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. ab236872 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.

Immunohistochemical analysis of paraffin-embedded human testis tissue labeling PBK/SPK with ab236872 at 1/4000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). 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 a ready to use Goat Anti-Rabbit IgG H&L (HRP).
Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

All lanes : Anti-PBK/SPK antibody [EPR21983] (ab236872) at 1/1000 dilution

Lane 1 : SW480 (human colorectal adenocarcinoma epithelial cell), whole cell lysate
Lane 2 : MCF7 (human breast adenocarcinoma epithelial cell), whole cell lysate
Lane 3 : A431 (human epidermoid carcinoma epithelial cell), whole cell lysate
Lane 4 : U-87 MG (human glioblastoma-astrocytoma epithelial cell), whole cell lysate
Lane 5 : Hepa1-6 (mouse hepatoma epithelial cell), whole cell lysate
Lane 6 : Human testis lysate
Lane 7 : Rat testis lysate
Lane 8 : C6 (rat glial tumor glial cell), whole cell lysate
Lane 9 : PC-12 (rat adrenal gland pheochromocytoma), whole cell lysate
Lane 10 : RAW 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage), whole cell lysate

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?

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure times: Lanes 1-4 & 8-9: 15 seconds; Lanes 5 & 10: 3 minutes; Lane 6: 70 seconds; Lane 7: 3 seconds.

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

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cell line labeling PBK/SPK with ab236872 at 1/500 (red) compared with a Rabbit monoclonal IgG (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077), at 1/2000 dilution was used as the secondary antibody.

PBK/SPK was immunoprecipitated from 0.35 mg HepG2 (human hepatocellular carcinoma epithelial cell) whole cell lysate with ab236872 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab236872 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution.
Lane 1: HepG2 whole cell lysate 10 µg (Input).
Lane 2: ab236872 IP in HepG2 whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab236872 in HepG2 whole cell lysate.
Blocking/Dilution buffer: 5% NFDM/TBST.
Exposure time: 10 seconds.

Immunohistochemical analysis of paraffin-embedded rat testis tissue labeling PBK/SPK with ab236872 at 1/4000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining in germ cells of rat testis (PMID:16982762; PMID:25909225) is observed. Counterstained with hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).
Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Immunohistochemical analysis of paraffin-embedded mouse testis tissue labeling PBK/SPK with ab236872 at 1/4000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Positive staining in germ cells of mouse testis (PMID:16982762; PMID:25909225) is observed. Counterstained with hematoxylin.
Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).
Perform 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 ab236872 at 1/4000 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). 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 a ready to use Goat Anti-Rabbit IgG H&L (HRP).
Perform heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized HepG2 (human hepatocellular carcinoma epithelial cell) cell line labeling PBK/SPK with ab236872 at 1/500 (red) compared with a Rabbit monoclonal IgG (ab172730) (black) and an unlabeled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077), at 1/2000 dilution was used as the secondary antibody.