Recombinant Anti-PKC delta antibody [EPR17075] (ab182126)

Lanes 1- 2: Merged signal (red and green). Green - ab182126 observed at 80 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab182126 was shown to react with PKC delta in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265721 (knockout cell lysate ab257043) was used. Wild-type HeLa and PKC delta knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab182126 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) overnight at 4°C at a 1 in 5000 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.

ab182126 staining PKCδ in untreated wild-type HAP1 cells (top panel) and PKCδ untreated knockout HAP1 cells (bottom panel). Untreated cells show PKCδ being expressed in the cytoplasm. The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab182126 at 1/200 dilution and ab7291 at 1ug/ml concentration overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor^® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to Mouse IgG (Alexa Fluor^® 594) (ab150117) at 2ug/ml (shown in pseudo-color red). Nuclear DNA was labelled in blue with DAPI.

This product also gave a positive signal under the same testing conditions in HAP1 cells fixed with 100% methanol (5 min).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Immunohistochemical analysis of paraffin-embedded rat testis tissue labeling PKC delta with ab182126 at 1/2000 dilution, followed by Anti-Rabbit HRP (ab97051) at 1/500 dilution. Cytoplasmic and weak nuclear staining on cells in the seminiferous tubule of rat testis is detected. The negative control utilised PBS instead of primary antibody. Counter stained with Hematoxylin.

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

Lanes 1 - 4: Merged signal (red and green). Green - ab182126 observed at 78 kDa. Red - loading control, ab8226, observed at 42 kDa.

ab182126 was shown to specifically react with PKC delta when PKC delta knockout samples were used. Wild-type and PKC delta knockout samples were subjected to SDS-PAGE. ab182126 and ab8226 (loading control to beta Actin) were diluted to 1/5000 and 1/1000 respectively and incubated overnight at 4°C. 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 1/10 000 dilution for 1 h at room temperature before imaging.

Lanes 1 - 2: Merged signal (red and green). Green - ab182126 observed at 78 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab182126 was shown to react with PKC in wild-type HEK-293T cells in western blot. The bands observed in PRKCD CRISPR/Cas9 edited cell line ab266143 (PRKCD CRISPR/Cas9 edited cell lysate ab257042) below 78kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type and PRKCD CRISPR/Cas9 edited HEK-293T cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab182126 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4

176;® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Intracellular Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling PKC delta with purified ab182126 at 1/250 dilution (10ug/mL) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cellswithout incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control. 

ab182126 staining PKCδ in 10nM PMA-treated wild-type HAP1 cells (top panel) and PKCδ in 10nM PMA-treated knockout HAP1 cells (bottom panel). The cells were treated with 10nM PMA for 10 minutes to induce translocation of PKCδ to the cell membrane. The cells were then fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab182126 at 1/200 dilution and ab7291 at 1ug/ml concentration overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor^® 488) (ab150081) at 2 μg/ml (shown in green) and a goat secondary antibody to Mouse IgG (Alexa Fluor^® 594) (ab150117) at 2ug/ml (shown in pseudo-color red). Nuclear DNA was labelled in blue with DAPI.

This product also gave a positive signal under the same testing conditions in HAP1 cells fixed with 100% methanol (5 min).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

The 40kDa band represents the cleaved kinase domain.

Blocking/dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labeling PKC delta with ab182126 at 1/2000 dilution, followed by Anti-Rabbit HRP (ab97051) at 1/500 dilution. Cytoplasmic and nuclear staining on kupffer cells of Mouse liver is detected. The negative control utilised PBS instead of primary antibody. Counter stained with Hematoxylin.

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

The 40kDa band represents the cleaved kinase domain.

Blocking/dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded Human transitional cell carcinoma of bladder tissue labeling PKC delta with ab182126 at 1/2000 dilution, followed by Anti-Rabbit HRP (ab97051) at 1/500 dilution. Cytoplasmic and weak nuclear staining on cancer cells of bladder transitional cell carcinoma is detected. The negative control utilised PBS instead of primary antibody. Counter stained with Hematoxylin.

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

Immunofluorescent analysis of 4% paraformaldehyde-fixed HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling PKC delta with ab182126 at 1/100 dilution. The cells were permeabilised with 0.1% Triton X-100. Goat anti-rabbit IAlexa Fluor^® 488 (IgG) (ab150077) at 1/400 dilution was used as the secondary antibody (green). The confocal image shows both cytoplasmic and nuclear staining on HeLa cells. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/500 and ab150120 (goat anti-mouse AlexaFluor^® 594 secondary antibody) at 1/500 dilution (red).
The negative controls are as follows;
1. ab182126 at 1/100 dilution followed by ab150120 (Alexa Fluor^® 594 Goat anti-Mouse secondary) at 1/500 dilution.
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.

The 40kDa band represents the cleaved kinase domain.

Blocking/dilution buffer: 5% NFDM/TBST.

Immunohistochemical analysis of paraffin-embedded Human spleen tissue labeling PKC delta with ab182126 at 1/2000 dilution, followed by Anti-Rabbit HRP (ab97051) at 1/500 dilution. Cytoplasmic and nucleus staining on lymphocytes of Human spleen is detected. The negative control utilised PBS instead of primary antibody. Counter stained with Hematoxylin.

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

The 40kDa band represents the cleaved kinase domain.

Blocking/dilution buffer: 5% NFDM/TBST.