Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HEK293T (Human epithelial cells from embryonic kidney) cells transfected with Empty vector or Firefly Luciferase, labeling Firefly Luciferase with ab185924 at 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/500 dilution (green). Nuclear staining on HEK293T cells transfected with Firefly Luciferase is observed. The nuclear counter stain is DAPI (blue). Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution (red).
The negative controls are as follows;
-ve control 1: ab185924 at 1/500 dilution followed by ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/500 dilution.
-ve control 2: ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor®488 Goat Anti-Rabbit IgG H&L) at 1/500 dilution.

Flow Cytometry analysis of 293T (Human epithelial cell line from embryonic kidney) cells transfected with GFP tagged Firefly Luciferase labeled with purified ab185924 at 1/70 dilution (10µg/ml, Right panel). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 647)(1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Left panel) was used as the isotype control.

All lanes : Anti-Firefly Luciferase antibody [EPR17790] (ab185924) at 1/20000 dilution

Lane 1 : Empty vector (vector control) transfected HEK293 (Human embryonic kidney) whole cell lysate
Lane 2 : Firefly Luciferase transfected HEK293 (Human embryonic kidney) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugate at 1/1000 dilution

Predicted band size: 61 kDa
Observed band size: 61 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-Firefly Luciferase antibody [EPR17790] (ab185924) at 1/1000 dilution

Lane 1 : Firefly Luciferase transfected HEK293 (human embryonic kidney) whole cell lysate at 10 µg
Lane 2 : HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate at 20 µg
Lane 3 : MCF7 (Human breast adenocarcinoma cell line) whole cell lysate at 20 µg
Lane 4 : Jurkat (Human T cell leukemia cells from peripheral blood) whole cell lysate at 20 µg
Lane 5 : SH-SY5Y (Human neuroblastoma from bone marrow cells) whole cell lysate at 20 µg
Lane 6 : Human fetal brain lysate at 10 µg
Lane 7 : Human fetal heart lysate at 10 µg
Lane 8 : Human fetal kidney lysate at 10 µg
Lane 9 : Human fetal spleen lysate at 10 µg

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/1000 dilution

Predicted band size: 61 kDa
Observed band size: 61 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

All lanes : Anti-Firefly Luciferase antibody [EPR17790] (ab185924) at 1/1000 dilution

Lane 1 : Firefly Luciferase transfected HEK293 (Human embryonic kidney) whole cell lysate
Lane 2 : Mouse brain lysate
Lane 3 : Mouse heart lysate
Lane 4 : Mouse kidney lysate
Lane 5 : Mouse spleen lysate
Lane 6 : Rat brain lysate
Lane 7 : Rat heart lysate
Lane 8 : Rat kidney lysate
Lane 9 : Rat spleen lysate
Lane 10 : C6 (Rat glial tumor cells) whole cell lysate
Lane 11 : RAW 264.7 (Mouse macrophage cells transformed with Abelson murine leukemia virus) whole cell lysate
Lane 12 : PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysate
Lane 13 : NIH/3T3 (Mouse embyro fibroblast cells) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugate at 1/1000 dilution

Predicted band size: 61 kDa
Observed band size: 61 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.