Anti-Gephyrin antibody [EPR12651(B)] - BSA and Azide free

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-Gephyrin antibody [EPR12651(B)] - BSA and Azide free (AB249939)

This data was developed using ab177154, the same antibody clone in a different buffer formulation.

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

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Gephyrin antibody [EPR12651(B)] - BSA and Azide free (AB249939)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab177154).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Gephyrin antibody [EPR12651(B)] - BSA and Azide free (AB249939)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab177154).

• WB

Supplier Data

Western blot - Anti-Gephyrin antibody [EPR12651(B)] - BSA and Azide free (AB249939)

This data was developed using ab177154, the same antibody clone in a different buffer formulation.

All lanes:

Western blot - Anti-Gephyrin antibody [EPR12651(B)] - BSA and Azide free (ab249939) at 1/1000 dilution

Lane 1:

Human fetal brain lysate at 10 µg

Lane 2:

293T lysate at 10 µg

Lane 3:

SH-SY5Y lysate at 10 µg

Lane 4:

Jurkat 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

• WB

Lab

Western blot - Anti-Gephyrin antibody [EPR12651(B)] - BSA and Azide free (AB249939)

This data was developed using ab177154, the same antibody clone in a different buffer formulation.

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)] - BSA and Azide free (ab249939) 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

false