All lanes : Anti-PKC (phospho T514) antibody [EP2730Y] (ab109539) at 1/2000 dilution (purified)

Lane 1 : Rat brain lysates
Lane 2 : Rat brain lysates and then the membrane was incubated with phosphatase.

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: 78 kDa



Blocking and diluting buffer : 5% NFDM/TBST

ab109539 (purified) at 1:20 dilution (2µg) immunoprecipitating PKC in Rat brain lysate.
Lane 1 (input): Rat brain lysate 10µg
Lane 2 (+): ab109539 & Rat brain lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab109539 in Rat brain lysate
For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:1000 dilution.
Blocking and diluting buffer: 5% NFDM/TBST.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Rat cerebellum tissue sections labeling PKC with Purified ab109539 at 1:300 dilution (1.14 µg/ml). Heat mediated antigen retrieval was performed using citrate buffer, pH 6.0. Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Mouse cerebellum tissue sections labeling PKC with Purified ab109539 at 1:300 dilution (1.14 µg/ml). Heat mediated antigen retrieval was performed using citrate buffer, pH 6.0. Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human cerebral cortex tissue sections labeling PKC with Purified ab109539 at 1:300 dilution (1.14 µg/ml). Heat mediated antigen retrieval was performed using citrate buffer, pH 6.0. Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1:0 dilution. PBS instead of the primary antibody was used as the negative control.

All lanes : Anti-PKC (phospho T514) antibody [EP2730Y] (ab109539) at 1/2000 dilution (purified)

Lane 1 : Mouse cerebellum lysates
Lane 2 : Mouse cerebellum lysates and then the membrane was incubated with phosphatase.

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: 78 kDa



Blocking and diluting buffer : 5% NFDM/TBST

All lanes : Anti-PKC (phospho T514) antibody [EP2730Y] (ab109539) at 1/2000 dilution (unpurified)

Lane 1 : Active human PKC alpha full length protein.
Lane 2 : Active human PKC beta 1 full length protein.
Lane 3 : Active human PKC beta 2 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.

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

Predicted band size: 78 kDa
Observed band size: 100-150 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking and Diluting buffer and concentration

Active human PKC alpha full length protein (Catalog#ab55672) contains aa1-672 with GST-tag; 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 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 (phospho T514) antibody [EP2730Y] (ab109539) at 1/5000 dilution (purified)

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates
Lane 2 : HeLa (Human cervix adenocarcinoma epithelial cell) treated with Phorbol-12-myristate-13-acetate whole cell lysates
Lane 3 : HeLa (Human cervix adenocarcinoma epithelial cell) treated with Phorbol-12-myristate-13-acetate whole cell lysates. The membrane was then incubated with phosphatase.

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: 78 kDa
Observed band size: 80 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking and dilution buffer: 5% NFDM/TBST.

All lanes : Anti-PKC (phospho T514) antibody [EP2730Y] (ab109539) at 0.02 µg/ml (unpurified)

Lane 1 : HeLa cell lysate with PKC gamma (pT514) peptide
Lane 2 : HeLa cell lysate with PKC gamma non-phospho peptide
Lane 3 : HeLa cell lysate

Predicted band size: 78 kDa
Observed band size: 80 kDa why is the actual band size different from the predicted?


Exposure time: 15 seconds

Unpurified ab109539 at 1/100 dilution staining PKC (phospho T514) in paraffin-embedded Human brain tissue by Immunohistochemistry.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.