Anti-14-3-3 antibody (ab9063)

  Western blot - 14-3-3 antibody (ab9063)

All lanes : Anti-14-3-3 antibody (ab9063) at 1/1000 dilution

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : Lung (Human) Tissue Lysate
Lane 3 : Brain (Human) Tissue Lysate - adult normal tissue (ab29466)

Lysates/proteins at 10 µg per lane.

Secondary
Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

  Immunoprecipitation - 14-3-3 antibody (ab9063)

14-3-3 is required for CT-Raf kinase activity. 14-3-3 can be removed from CT-Raf in vitro with the detergent Empigen-BB. Sf9 cells were infected with recombinant baculoviruses encoding either GST or a GST–CT-Raf fusion protein. Forty-eight hours after infection, the GST or GST–CT-Raf proteins were isolated from NP-40 lysates by using glutathione-agarose beads.

The bead-bound complexes were then washed with either NP-40 lysis buffer (lanes 1 and 2) or NP-40 lysis buffer containing 1% Empigen-BB (lane 3), resolved by SDS-PAGE, transferred to nitrocellulose, and developed with an antibody to either 14-3-3 (upper panel) or Raf (lower panel).

Taken from Thorsen, J.A., et al, (1998) with permission

  Immunocytochemistry/ Immunofluorescence-14-3-3 antibody(ab9063)

ICC/IF image of ab9063 stained PC12 cells. The cells were 100% methanol fixed (5 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 (ab9063, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 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.