All lanes : Anti-PKC antibody [EPR16898] (ab179522) at 1/2000 dilution

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 gamma full length protein
Lane 5 : Active human PKC delta full length protein
Lane 6 : Active human PKC eta full length protein
Lane 7 : Active human PKC epsilon full length protein
Lane 8 : Active human PKC theta full length protein
Lane 9 : 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)

Predicted band size: 77 kDa


Exposure time: 2 seconds

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 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.

Blocking/Diluting buffer 5% NFDM/TBST

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized K562 (Human chronic myelogenous leukemia cells from bone marrow) cells labeling PKC alpha + beta + gamma with ab179522 at 1/200 dilution, followed by Goat anti-rabbit IAlexa Fluor® 488 (IgG) (ab150077) secondary antibody at 1/400 dilution (green). Confocal image shows cytoplasmic staining on K562 cell line. The nuclear counterstain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution and ab150120 (goat anti-mouse AlexaFluor®594 secondary antibody) at 1/500 dilution (red).

The negative controls are as follows:-
-ve control 1: - ab179522 at 1/200 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2: - ab7291 (anti-Tubulin mouse mAb) at 1/500 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/400 dilution.

All lanes : Anti-PKC antibody [EPR16898] (ab179522) at 1/2000 dilution

Lane 1 : Active human PKC zeta full length protein
Lane 2 : 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: 77 kDa


Exposure time: 3 minutes

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.

Blocking/Diluting buffer 5% NFDM/TBST

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

All lanes : Anti-PKC antibody [EPR16898] (ab179522) at 1/10000 dilution

Lane 1 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate at 20 µg
Lane 2 : 293T (Human epithelial cells from embryonic kidney) whole cell lysate at 10 µg
Lane 3 : SH-SY5Y (Human neuroblastoma from bone marrow cells) whole cell lysate at 10 µg

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

Predicted band size: 77 kDa
Observed band size: 77 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-PKC antibody [EPR16898] (ab179522) at 1/1000 dilution

Lane 1 : K562 (Human chronic myelogenous leukemia cells from bone marrow) whole cell lysate at 20 µg
Lane 2 : A431 (Human epidermoid carcinoma) whole cell lysate at 10 µg

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

Predicted band size: 77 kDa
Observed band size: 77 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-PKC antibody [EPR16898] (ab179522) at 1/1000 dilution

Lane 1 : Human fetal brain lysate
Lane 2 : Human fetal heart lysate
Lane 3 : Human fetal kidney 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: 77 kDa
Observed band size: 77 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-PKC antibody [EPR16898] (ab179522) at 1/5000 dilution

Lane 1 : L6 (Rat skeletal muscle cell line) whole cell lysate
Lane 2 : Neuro-2a (Mouse neuroblastoma cells) whole cell lysate
Lane 3 : L929 (Mouse connective tissue fibroblast cells) 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: 77 kDa
Observed band size: 77 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

Anti-PKC antibody [EPR16898] (ab179522) at 1/1000 dilution + Rat brain whole cell lysate at 10 µg

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

Predicted band size: 77 kDa
Observed band size: 77 kDa

Blocking/Dilution buffer: 5% NFDM/TBST.

Flow cytometric analysis of 2% paraformaldehyde-fixed NIH/3T3 (Mouse embyro fibroblast cells) cells labeling PKC alpha + beta + gamma with ab179522 at 1/360 dilution (red) compared with a rabbit monoclonal IgG isotype control (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 alpha + beta + gamma was immunoprecipitated from 1mg of Rat brain extract with ab179522 at 1/100 dilution. Western blot was performed from the immunoprecipitate using ab179522 at 1/1000 dilution. Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated antibody was used as secondary antibody at 1/1000 dilution.

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