All lanes : Anti-PKC antibody [EPR17368] (ab181558) at 1/2000 dilution

Lane 1 : Active human PKC beta 1 full length protein
Lane 2 : Active human PKC beta 2 full length protein
Lane 3 : Active human PKC gamma full length protein
Lane 4 : Active human PKC delta full length protein
Lane 5 : Active human PKC eta full length protein
Lane 6 : Active human PKC epsilon full length protein
Lane 7 : Active human PKC theta full length protein
Lane 8 : Active human PKC mu full length protein

Lysates/proteins at 0.02 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 68 kDa


Exposure time: 2 seconds

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure time:2 seconds

Active human PKC beta 1 full length protein (Catalog#ab60840) contains aa1-671 with GST-tag; Active human PKC beta 2 full length protein (Catalog#ab60841) contains aa1-673 with GST-tag; Active human PKC gamma full length protein (Catalog#ab60842) contains aa1-677 with GST-tag; Active human PKC delta full length protein (Catalog#ab60844) contains aa1-676 with GST-tag; Active human PKC eta full length protein (Catalog#ab60849) contains aa1-683 with GST-tag; Active human PKC epsilon full length protein (Catalog#ab60847) contains aa1-737 with GST-tag; Active human PKC theta full length protein (Catalog#ab56641) contains aa1-706 with GST-tag; Active human PKC mu full length protein (Catalog#ab60873) contains aa1-912 with GST-tag.

All lanes : Anti-PKC antibody [EPR17368] (ab181558) at 1/2000 dilution

Lane 1 : Active human PKC alpha full length protein
Lane 2 : Active human PKC zeta full length protein
Lane 3 : Active human PKC iota full length protein

Lysates/proteins at 0.02 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Predicted band size: 68 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure times: Lane 1:  10 seconds; Lane 2 and 3: 3 seconds.

Active human PKC alpha full length protein (Catalog#ab55672) contains aa1-672 with GST-tag; Active human PKC zeta full length protein (Catalog# ab60848) contains aa1-592 with GST-tag; Active human PKC iota full length protein (Catalog#ab60850) contains aa1-596 with GST-tag.

Recombinant PKC isoforms were tested for reactivity by western blot, and are marked as positive (+) or negative (-).

All lanes : Anti-PKC antibody [EPR17368] (ab181558) at 1/2000 dilution

Lane 1 : A431 (Human epidermoid carcinoma cell line) whole cell lysate
Lane 2 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 3 : 293T (Human epithelial cell line from embryonic kidney) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 68 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-PKC antibody [EPR17368] (ab181558) at 1/10000 dilution

Lane 1 : Human fetal kidney lysate
Lane 2 : Human fetal brain lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 68 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-PKC antibody [EPR17368] (ab181558) at 1/10000 dilution

Lane 1 : Mouse spleen lysate
Lane 2 : Rat spleen lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 68 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-PKC antibody [EPR17368] (ab181558) at 1/2000 dilution

Lane 1 : C6 (Rat glial tumor cell line) whole cell lysate
Lane 2 : RAW 264.7 (Mouse macrophage cell line transformed with Abelson murine leukemia virus) whole cell lysate
Lane 3 : PC-12 (Rat adrenal gland pheochromocytoma cell line) whole cell lysate
Lane 4 : NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 68 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-PKC antibody [EPR17368] (ab181558) at 1/50000 dilution

Lane 1 : Rat pancreas lysate
Lane 2 : Mouse pancreas lysate
Lane 3 : Mouse lung lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L),Peroxidase conjugated at 1/1000 dilution

Predicted band size: 68 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labeling PKC (various isoforms) with ab181558 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Cytoplasmic and weak nuclear staining on glomerular and renal tubule is observed.

Reference:

1: Y Mizukam,T Hirata,et.al.(1997) Nuclear translocation of PKC zeta during ischemia and its inhibition by wortmannin, an inhibitor of phosphatidylinositol

2: Jose A. Crespo,Petra Stockl,et.al(2012) Activation of PKCzeta and PKMzeta in the Nucleus Accumbens Core Is Necessary for the Retrieval,Consolidation and Reconsolidation of Drug Memory.Plos One 7(2):e30502.

3-kinase.FEBS Lett 401 (2-3): 247-251.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Human gastric adenocarcinoma tissue labeling PKC (various isoforms) with ab181558 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Cytoplasmic and weak nuclear staining on Human gastric adenocarcinoma is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Mouse testis tissue labeling PKC (various isoforms) with ab181558 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Cytoplasmic staining on testis seminiferous tubule is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded Rat kidney tissue labeling PKC (various isoforms) with ab181558 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Cytoplasmic and weak nuclear staining on glomerular and renal tubule is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab97051 at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized A431 (Human epidermoid carcinoma cell line) cells labeling PKC (various isoforms) with ab181558 at 1/500 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/400 dilution (green).

Confocal image showing cytoplasm staining on A431 cell line. The nuclear counter stain is DAPI (blue).

Tubulin is detected with Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) at 1/1000 dilution and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) at 1/1000 dilution (red).

The negative controls are as follows:
-ve control 1: ab181558 at 1/500 dilution followed by ab150120 at 1/500 dilution.
-ve control 2: ab7291  at 1/500 dilution followed by ab150077 at 1/400 dilution.

Flow cytometric analysis of 2% paraformaldehyde-fixed NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling PKC (various isoforms) with ab181558 at 1/410 dilution (red) compared with a Rabbit IgG,monoclonal[EPR25A]-Isotype control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). Goat anti Rabbit IgG (FITC) at 1/150 dilution was used as the secondary antibody.

PKC was immunoprecipitated from 1 mg of A431 (Human epidermoid carcinoma cell line) whole cell extract with ab181558 at 1/100 dilution. Western blot was performed from the immunoprecipitate using ab181558 at 1/5000 dilution. Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG was used as secondary antibody at 1/1500 dilution.

Lane 1: A431 whole cell extract.

Lane 2: PBS.

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