Anti-GEF H1 antibody [EPR17963] - BSA and Azide free

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-GEF H1 antibody [EPR17963] - BSA and Azide free (AB251352)

This data was developed using ab201687, the same antibody clone in a different buffer formulation.

ab201687 was shown to react with ARHGEF2 in wild-type U-87 MG cells in immunocytochemistry with loss of signal observed in a ARHGEF2 siRNA knockdown cell line. Wild-type and siRNA knockdown cells were mixed and pelleted at a 1 : 1 ratio on coverslips. The cells were fixed with 4% paraformaldehyde (15 min) then permeabilized with 0.1% Triton X-100 (10min) and then blocked with 1x PBS, 0.01% Triton X-100, 5% BSA, 5% NGS. The cells were then incubated with ab201687 at 1/500 dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat anti-rabbit secondary antibody to (Alexa Fluor^® 555) at 0.5 μg/ml. Acquisition of the green (wild-type), red (antibody staining) and far-red (siRNA knockdown) channels was performed. Representative grayscale images of the red channel are shown. Wild-type and siRNA knockdown cells are outlined with yellow and magenta dashed line, respectively. Schematic representation of the mosaic strategy used is shown on the bottom-right panel. Image was acquired with a Zeiss (LSM-880).

This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

• IP

Supplier Data

Immunoprecipitation - Anti-GEF H1 antibody [EPR17963] - BSA and Azide free (AB251352)

This data was developed using ab201687, the same antibody clone in a different buffer formulation.

Immunoprecipitation of ARHGEF2 in U-87 MG cells. Lysates were prepared and immunoprecipitation was performed using 2 μg of ab201687 pre-coupled to Protein A beads. Samples were then washed and processed for western blot.

This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Immunoprecipitation - Anti-GEF H1 antibody [EPR17963] - C-terminal (<a href='/en-us/products/primary-antibodies/gef-h1-antibody-epr17963-c-terminal-ab201687'>ab201687</a>) at 2 µg

All lanes:

U-87 MG cell lysates

Observed band size: 111.5 kDa

false

• WB

Supplier Data

Western blot - Anti-GEF H1 antibody [EPR17963] - BSA and Azide free (AB251352)

This data was developed using ab201687, the same antibody clone in a different buffer formulation.

ab201687 was shown to react with ARHGEF2 in wild-type U-87 MG cells in Western blot with loss of signal observed in a ARHGEF2 siRNA knockdown cell line. Cell lysates from wild-type U-87 MG transfected with either scrambled siRNA or ARHGEF2 siRNA were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab201687 overnight at 4 °C at a 1/2000 dilution. Blots were incubated with secondary antibodies at 0.2 µg/mL before imaging.

This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Western blot - Anti-GEF H1 antibody [EPR17963] - C-terminal (<a href='/en-us/products/primary-antibodies/gef-h1-antibody-epr17963-c-terminal-ab201687'>ab201687</a>) at 1/2000 dilution

Lane 1:

Wild-type U-87 MG transfected with scrambled siRNA control lysate at 30 µg

Lane 2:

U-87 MG transfected with siRNA specifically targeting ARHGEF2 cell lysate at 30 µg

Observed band size: 111.5 kDa

false

• WB

Lab

Western blot - Anti-GEF H1 antibody [EPR17963] - BSA and Azide free (AB251352)

This data was developed using the same antibody clone in a different buffer formulation (ab201687).

Lanes 1-3 : Merged signal (red and green). Green - ab201687 observed at 75 kDa. Red - loading control, ab8245 observed at 37 kDa.

ab201687 Anti-GEF H1 antibody [EPR17963] - C-terminal was shown to specifically react with GEF H1 in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab265797 (knockout cell lysate ab257223) was used. Wild-type and GEF H1 knockout samples were subjected to SDS-PAGE. ab201687 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-GEF H1 antibody [EPR17963] - C-terminal (<a href='/en-us/products/primary-antibodies/gef-h1-antibody-epr17963-c-terminal-ab201687'>ab201687</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

ARHGEF knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human ARHGEF2 (GEF H1) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-arhgef2-gef-h1-knockout-hela-cell-line-ab265797'>ab265797</a>)

Lane 3:

HEK-293T cell lysate at 20 µg

Predicted band size: 111 kDa

Observed band size: 112 kDa,75 kDa

false