Anti-p115-RhoGEF antibody [JH-1]

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-p115-RhoGEF antibody [JH-1] (AB243248)

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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-p115-RhoGEF antibody [JH-1] (AB243248)

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 Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-p115-RhoGEF antibody [JH-1] (AB243248)

Intracellular 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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-p115-RhoGEF antibody [JH-1] (AB243248)

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.

• ICC

Lab

Immunocytochemistry - Anti-p115-RhoGEF antibody [JH-1] (AB243248)

ICC/IF image of ab243248 stained TRA-differentiated 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 (orange) was ab175716 Goat Anti-Armenian hamster IgG H&L (Alexa Fluor^® 568) 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.

• WB

Lab

Western blot - Anti-p115-RhoGEF antibody [JH-1] (AB243248)

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.

All lanes:

Western blot - Anti-p115-RhoGEF antibody [JH-1] (ab243248) at 1/1000 dilution

Lane 1:

A20 (mouse reticulum sarcoma B lymphocyte), whole cell lysate at 10 µg

Lane 2:

EL4 (mouse lymphoma T lymphocyte), whole cell lysate at 10 µg

Lane 3:

PC-12 (rat adrenal gland pheochromocytoma), whole cell lysate at 10 µg

Lane 4:

C6 (rat glial tumor glial cell), whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Rabbit Anti-Armenian hamster IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/rabbit-armenian-hamster-igg-h-l-hrp-ab5745'>ab5745</a>) at 1/5000 dilution

Predicted band size: 102 kDa

Observed band size: 115 kDa

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