Immunofluorescent analysis of Red Firefly Luciferase (from the Japanese firefly Luciola cruciata) in pCMV-Red Firefly Luciferase transfected (green, right panel) and untransfected 293 cells (left panel). Formalin-fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 10 minutes at room temperature. Cells were probed with ab187340 at 1/1000 dilution for at least 1 hour at room temperature, washed with PBS, and incubated with a DyLight 488-conjugated goat anti-rabbit IgG secondary antibody at 1/400 dilution for 30 minutes at room temperature. Nuclei (blue) were stained with Hoechst 33342 dye.

All lanes : Anti-Firefly Luciferase antibody (ab187340) at 1/4000 dilution

Lane 1 : Non-transfected 293 whole cell lysate
Lane 2 : Green Renilla Luciferase transfected 293 whole cell lysate
Lane 3 : Red Firefly Luciferase transfected 293 whole cell lysate

Blocking peptides at 10 µg per lane.

Secondary
All lanes : Goat anti-rabbit HRP at 1/500 dilution

Developed using the ECL technique.

Predicted band size: 60 kDa

All lanes : Anti-Firefly Luciferase antibody (ab187340) at 1/4000 dilution

Lane 1 : Non-transfected 293 whole cell lysate
Lane 2 : Red Firefly Luciferase transfected 293 whole cell lysate
Lane 3 : North American firefly (Photynus pyralis) Luciferase transfected 293 whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat anti-rabbit HRP at 1/500 dilution

Developed using the ECL technique.

Predicted band size: 60 kDa

All lanes : Anti-Firefly Luciferase antibody (ab187340) at 1/4000 dilution

Lane 1 : Full length recombinant Red Firefly Luciferase at 100 µg
Lane 2 : Full length recombinant Red Firefly Luciferase at 50 µg
Lane 3 : Full length recombinant Red Firefly Luciferase at 25 µg
Lane 4 : Full length recombinant Red Firefly Luciferase at 10 µg

Secondary
All lanes : Goat anti-rabbit HRP at 1/500 dilution

Developed using the ECL technique.

Predicted band size: 60 kDa

Immunoprecipitation of Red Firefly Luciferase (from the Japanese firefly Luciola cruciata) was performed using whole cell lysates  from untransfected and Red Firefly or Green Renilla Luciferase transfected 293 cell lines. Antigen-antibody complexes were formed by incubating 200µg of lysate with 5µg of ab187340 overnight on a rocking platform at 4°C. The immune complexes were captured on 50ul Protein A/G Agarose, washed extensively, and eluted. Whole cell lysates from untransfected and transfected whole cell lysates (2µg) were used as controls. Samples were resolved on a 4-20% Tris-HCl polyacrylamide gel, transferred to a PVDF membrane, and blocked with 5% BSA/TBS-0.1%Tween-20 for 1 hour. The membrane was probed with ab187340 at 1/4000 dilution overnight rotating at 4°C.

 

Lane 1: Non-transfected 293 whole cell lysate

Lane 2: IP using ab187340 from non-transfected 293 whole cell lysate

Lane 3: Green Renilla Luciferase transfected 293 whole cell lysate

Lane 4: IP using ab187340 from Green Renilla Luciferase transfected 293 whole cell lysate

Lane 5: Red Firefly Luciferase transfected 293 whole cell lysate
Lane 6: IP using ab187340 from Red Firefly Luciferase transfected 293 whole cell lysate

Neutralizing effect of ab187340 on the activity of purified recombinant Red Firefly Luciferase (from the Japanese firefly Luciola cruciata). 10ng of recombinant enzyme were incubated with the indicated amounts of ab187340 for at least 30 min. Enzyme activity (RLU) was detected. Average RLU values from 10 timepoints per concentration of 4 replicates +/- standard deviation is shown.