Anti-Protein Kinase D2抗体(ab7281)

ab7281 staining Protein Kinase D2 in human BON cells (derived from a human pancreatic carcinoid tumor) by Immunocytochemistry/ Immunofluorescence.
BON cells were grown on glass coverslips in 24-well plates. 2 or 3 days after seeding or transfection, cells were treated with vehicle (Me₂SO) or PMA (100 nm) for 30 minutes. After treatment, cells were fixed with 4% paraformaldehyde for 20 minutes at 37 °C. After three washes with PBS, the cells were permeabilized with 0.3% Triton X-100 for 15 minutes at 37 °C and blocked with 1% bovine serum albumin/PBS for 10 minutes. The cells were incubated with the primary antibody diluted with 1% bovine serum albumin/PBS for 1 hour at room temperature. Cells were washed three times with PBS and incubated with Alexa-conjugated secondary antibody diluted by 1/500 in 1% bovine serum albumin/PBS.

Double staining was performed using antibodies ab7281 and an anti-NT. Co-localization is shown by arrows in the merged images.

Anti-Protein Kinase D2 antibody (ab7281) at 1/500 dilution + whole cell lysate prepared from murine NIH3T3 cells at 25 µg

Secondary
Donkey anti-rabbit HRP at 1/5000 dilution

Developed using the ECL technique.

Exposure time: 15 minutes

ICC/IF image of ab7281 stained HeLa cells. The cells were 4% formaldehyde (10 min) 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 (ab7281, 5µg/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.

Protein Kinase D2 was immunoprecipitated using 0.5mg NIH3T3 whole cell extract, 5µg of Rabbit polyclonal to Protein Kinase D2 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, NIH3T3 whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab7281.
Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band: 101KDa: Protein Kinase D2