ICC/IF image of ab243248 stained Neuro2a cells. The cells were 4% formaldehyde fixed (10 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then incubated in 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab243248, 1.6µg/ml) overnight at +4°C. The secondary antibody (green) was ab173003 Goat Anti-Armenian hamster IgG H&L (Alexa Fluor® 488) used at 1µg/ml for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

The negative control (inset) is a secondary-only assay to demonstrate low non-specific binding of the secondary antibody.

All lanes : Anti-p115-RhoGEF antibody [JH-1] (ab243248) at 1/1000 dilution

Lane 1 : A20 (mouse reticulum sarcoma B lymphocyte), whole cell lysate
Lane 2 : EL4 (mouse lymphoma T lymphocyte), whole cell lysate
Lane 3 : PC-12 (rat adrenal gland pheochromocytoma), whole cell lysate
Lane 4 : C6 (rat glial tumor glial cell), whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Rabbit Anti-Armenian hamster IgG H&L (HRP) (ab5745) at 1/5000 dilution

Predicted band size: 102 kDa
Observed band size: 115 kDa why is the actual band size different from the predicted?

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID: 11384980).

Lysates should be made freshly and used in WB immediately.

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure times: Lanes 1/2:3 seconds; Lane 3:15 seconds; Lane 4:37 seconds.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized A20 (mouse reticulum sarcoma B lymphocyte) cells labelling p115-RhoGEF with ab243248 at 1/50 dilution, followed by ab173003 Goat Anti-Armenian hamster IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in A20 cells. ab179513 Anti-beta Tubulin rabbit monoclonal antibody was used to counterstain tubulin at 1/200 dilution, followed by ab150080 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 594) at a 1/1000 dilution (Red). The nuclear counterstain was DAPI (Blue).

Negative control 1: ab243248 at a 1/50 dilution followed by ab150080 at a 1/1000 dilution.

Negative control 2: ab179513 at a 1/200 dilution followed by ab173003 at a 1/1000 dilution.

 

Flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized EL-4 (mouse lymphoma T lymphocyte) cells labelling p115-RhoGEF with ab243248 at 1/40 dilution (1µg) (Red) compared with an Armenian hamster monoclonal IgG (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti armenian hamster IgG (Alexa Fluor^® 488, ab173003) at 1/2000 dilution was used as the secondary antibody.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized EL4 (mouse lymphoma T lymphocyte) cells labelling p115-RhoGEF with ab243248 at 1/50 dilution, followed by ab173003 Goat Anti-Armenian hamster IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in EL4 cells. ab179513 Anti-beta Tubulin rabbit monoclonal antibody was used to counterstain tubulin at 1/200 dilution, followed by ab150080 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 594) at a 1/1000 dilution (Red). The nuclear counterstain was DAPI (Blue).

Negative control 1: ab243248 at a 1/50 dilution followed by ab150080 at a 1/1000 dilution.

Negative control 2: ab179513 at a 1/200 dilution followed by ab173003 at a 1/1000 dilution.