Anti-Gephyrin antibody [EPR12651(B)]

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-Gephyrin antibody [EPR12651(B)] (AB177154)

Intracellular flow cytometric analysis of permeabilized 293T cells labeling Gephyrinwith ab177154 at 1/10 dilution (red), or a rabbit IgG (negative) (green).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Gephyrin antibody [EPR12651(B)] (AB177154)

Immunohistochemical analysis of paraffin-embedded Human brain tissue labeling Gephyrin with ab177154 at 1/50 dilution.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Gephyrin antibody [EPR12651(B)] (AB177154)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Jurkat cells labelling Gephyrin with ab177154 at 1/100 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (2 μg/mL) (Green). Confocal image showing cytoplasmic staining in Jurkat cell line. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (2.5 μg/mL) (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : PBS instead of the primary antibody.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Gephyrin antibody [EPR12651(B)] (AB177154)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized PC-12 cells labelling Gephyrin with ab177154 at 1/100 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (2 μg/mL) (Green). Confocal image showing cytoplasmic staining in PC-12 cell line. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (2.5 μg/mL) (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : PBS instead of the primary antibody.

• WB

Lab

Western blot - Anti-Gephyrin antibody [EPR12651(B)] (AB177154)

Lanes 1 - 4 : Merged signal (red and green). Green - ab177154 observed at 90 kDa. Red - loading control, ab9484, observed at 37 kDa.

ab177154 was shown to recognize Gephyrin in wild-type HAP1 cells as signal was lost at the expected MW in GPHN (Gephyrin) knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and GPHN (Gephyrin) knockout samples were subjected to SDS-PAGE. ab177154 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1 μg/ml and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Gephyrin antibody [EPR12651(B)] (ab177154) at 1 µg/mL

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

GPHN (Gephyrin) knockout HAP1 whole cell lysate at 20 µg

Lane 3:

MCF7 whole cell lysate at 20 µg

Lane 4:

U2OS whole cell lysate at 20 µg

Predicted band size: 80 kDa

Observed band size: 90 kDa

false

• WB

Supplier Data

Western blot - Anti-Gephyrin antibody [EPR12651(B)] (AB177154)

All lanes:

Western blot - Anti-Gephyrin antibody [EPR12651(B)] (ab177154) at 1/1000 dilution

Lane 1:

Human fetal brain lysate at 10 µg

Lane 2:

293T lysate at 10 µg

Lane 3:

Jurkat lysate at 10 µg

Lane 4:

SH-SY5Y lysate at 10 µg

Lane 5:

C6 lysate at 10 µg

Lane 6:

Raw264.7 lysate at 10 µg

Lane 7:

PC-12 lysate at 10 µg

Secondary

All lanes:

Goat anti-rabbit HRP at 1/2000 dilution

Predicted band size: 80 kDa

false