Anti-PRKD2 antibody [EP1495Y]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-PRKD2 antibody [EP1495Y] (AB51250)

ab51250 (1 : 100) staining human PKD2 in human breast carcinoma tissue by immunohistochemistry using paraffin embedded tissue.

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

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-PRKD2 antibody [EP1495Y] (AB51250)

ICC/IF image of ab51250 stained HeLa cells. The cells were 4% Formaldehyde fixed (10 mins) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab51250 10μg/ml) overnight at +4°C. The secondary antibody (green) was ab96899 DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43μM.

• WB

Unknown

Western blot - Anti-PRKD2 antibody [EP1495Y] (AB51250)

All lanes:

Western blot - Anti-PRKD2 antibody [EP1495Y] (ab51250) at 1/500 dilution

All lanes:

HeLa cell lysate at 10 µg

Secondary

All lanes:

Goat anti-rabbit HRP labeled at 1/2000 dilution

Predicted band size: 97 kDa

Observed band size: 105 kDa

false

• WB

Lab

Western blot - Anti-PRKD2 antibody [EP1495Y] (AB51250)

Lane 1 : Wild-type HAP1 cell lysate (40 μg)
Lane 2 : PKD2 knockout HAP1 cell lysate (40 μg)
Lane 3 : HeLa cell lysate (20 μg)
Lane 4 : HEK293 cell lysate (20 μg)
Lanes 1 - 4 : Merged signal (red and green). Green - ab51250 observed at 110 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab51250 was shown to recognize PKD2 when PKD2 knockout samples were used, along with additional cross-reactive bands. Wild-type and PKD2 knockout samples were subjected to SDS-PAGE. ab51250 and ab8245 (loading control to GAPDH) were diluted at 1/500 and 1 : 10,000 dilution respectively and incubated overnight at 4C. 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 hour at room temperature before imaging.

All lanes:

Western blot - Anti-PRKD2 antibody [EP1495Y] (ab51250)

Predicted band size: 97 kDa

false

• WB

Lab

Western blot - Anti-PRKD2 antibody [EP1495Y] (AB51250)

Western blot : Anti-PRKD2 antibody [EP1495Y] (ab51250) staining at 1/500 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab51250 was shown to bind specifically to PRKD2. A band was observed at 105 kDa in wild-type A549 cell lysates with no signal observed at this size in PRKD2 knockout cell line. To generate this image, wild-type and PRKD2 knockout A549 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-PRKD2 antibody [EP1495Y] (ab51250) at 1/500 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

PRKD2 knockout A549 cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 105 kDa

false