Anti-PKC delta antibody [EPR17075] - BSA and Azide free

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-PKC delta antibody [EPR17075] - BSA and Azide free (AB222229)

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, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab182126).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PKC delta antibody [EPR17075] - BSA and Azide free (AB222229)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab182126).

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

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-PKC delta antibody [EPR17075] - BSA and Azide free (AB222229)

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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab182126).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-PKC delta antibody [EPR17075] - BSA and Azide free (AB222229)

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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab182126).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-PKC delta antibody [EPR17075] - BSA and Azide free (AB222229)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab182126).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PKC delta antibody [EPR17075] - BSA and Azide free (AB222229)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab182126).

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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PKC delta antibody [EPR17075] - BSA and Azide free (AB222229)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab182126).

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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PKC delta antibody [EPR17075] - BSA and Azide free (AB222229)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab182126).

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

• WB

Lab

Western blot - Anti-PKC delta antibody [EPR17075] - BSA and Azide free (AB222229)

This data was developed using ab182126, the same antibody clone in a different buffer formulation.

Western blot : Anti-PRKCD antibody [EPR17075] (ab182126) staining at 1/5000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab182126 was shown to bind specifically to PRKCD. A band was observed at 75-80 kDa in wild-type U-87 MG cell lysates with no signal observed at this size in PRKCD knockout cell line. To generate this image, wild-type and PRKCD knockout U-87 MG cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-PKC delta antibody [EPR17075] (<a href='/en-us/products/primary-antibodies/pkc-delta-antibody-epr17075-ab182126'>ab182126</a>) at 1/5000 dilution

Lane 1:

Wild-type U-87 MG cell lysate at 20 µg

Lane 2:

Western blot - Human PRKCD knockout U-87 MG cell line (<a href='/en-us/products/cell-lines/human-prkcd-knockout-u-87-mg-cell-line-ab306756'>ab306756</a>)

Lane 2:

PRKCD knockout U-87 MG cell lysate at 20 µg

Secondary

Lanes 1 - 2:

Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution

Lanes 1 - 2:

Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 75-80 kDa

false

• WB

Lab

Western blot - Anti-PKC delta antibody [EPR17075] - BSA and Azide free (AB222229)

This data was developed using the same antibody clone in a different buffer formulation (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 PRKCD 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.

All lanes:

Western blot - Anti-PKC delta antibody [EPR17075] (<a href='/en-us/products/primary-antibodies/pkc-delta-antibody-epr17075-ab182126'>ab182126</a>) at 1/5000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

PRKCD knockout HeLa cell lysate at 20 µg

Predicted band size: 78 kDa

Observed band size: 80 kDa

false

• WB

Lab

Western blot - Anti-PKC delta antibody [EPR17075] - BSA and Azide free (AB222229)

This data was developed using the same antibody clone in a different buffer formulation (ab182126).

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<sup>&

176;</sup>C at a 1 in 5000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 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.

All lanes:

Western blot - Anti-PKC delta antibody [EPR17075] (<a href='/en-us/products/primary-antibodies/pkc-delta-antibody-epr17075-ab182126'>ab182126</a>) at 1/5000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

PRKCD CRISPR/Cas9 edited HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human PRKCD (PKC delta) knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-prkcd-pkc-delta-knockout-hek-293t-cell-line-ab266143'>ab266143</a>)

Predicted band size: 78 kDa

Observed band size: 78 kDa

false

• WB

Lab

Western blot - Anti-PKC delta antibody [EPR17075] - BSA and Azide free (AB222229)

This data was developed using the same antibody clone in a different buffer formulation (ab182126).

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.

All lanes:

Western blot - Anti-PKC delta antibody [EPR17075] - BSA and Azide free (ab222229) at 1/5000 dilution

Lane 1:

Wild-type HAP1 cell lysate at 20 µg

Lane 2:

PKC delta knockout HAP1 cell lysate at 20 µg

Lane 3:

Jurkat cell lysate at 20 µg

Lane 4:

HeLa cell lysate at 20 µg

Predicted band size: 78 kDa

false