All lanes : Anti-BRG1 antibody [EPNCIR111A] (ab110641) at 1/10000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : SMARCA4 knockout HEK-293T cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 185 kDa
Observed band size: 185 kDa

Lanes 1- 2: Merged signal (red and green). Green - ab110641 observed at 185 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

 ab110641 was shown to react with SMARCA4 in wild-type HEK-293T cells in western blot. Loss of signal was observed when knockout cell line ab255432 (knockout cell lysate ab263853) was used. Wild-type HEK-293T and SMARCA4 knockout HEK-293T cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab110641 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) overnight at 4°C at a 1 in 10000 dilution and a 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 20000 dilution for 1 hour at room temperature before imaging.

ab110641 staining BRG1 in HeLa cells. The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab110641 at 1/500 dilution and ab195889 (Mouse monoclonal [DM1A] to alpha Tubulin - Microtubule Marker (Alexa Fluor® 594)) at 1/250 dilution overnight at +4°C, followed by a further incubation at room temperature for 1h with ab150081 (Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 488)) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Immunohistochemical analysis of paraffin-embedded Human cervical carcinoma tissue labeling BRG1 with ab110641, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Nuclear staining on human cervical carcinoma. The section was incubated with ab229902 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

All lanes : Anti-BRG1 antibody [EPNCIR111A] (ab110641) at 1/10000 dilution

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates
Lane 2 : NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates
Lane 3 : RAW 264.7(Mouse Abelson murine leukemia virus-induced tumor macrophage)whole cell lysates
Lane 4 : C6 (Rat glial tumor glial cell) whole cell lysates
Lane 5 : K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysates

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 185 kDa
Observed band size: 185 kDa

Blocking/Diluting buffer and concentration: 5% NFDM/TBST.

Exposure time: Lane 1 to 4: 5 seconds; Lane 5: 15 seconds

All lanes : Anti-BRG1 antibody [EPNCIR111A] (ab110641) at 1/10000 dilution

Lane 1 : Wild-type HAP1 cell lysate
Lane 2 : BRG1 knockout HAP1 cell lysate
Lane 3 : K562 cell lysate
Lane 4 : Molt-4 cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 185 kDa
Additional bands at: 185 kDa. We are unsure as to the identity of these extra bands.

Lanes 1 - 4: Merged signal (red and green). Green - ab110641 observed at 185 kDa. Red - loading control, ab18058, observed at 124 kDa.

ab110641 was shown to specifically react with BRG1 in wild-type HAP1 cells. No band was observed when BRG1 knockout samples were used. Wild-type and BRG1 knockout samples were subjected to SDS-PAGE, ab110641 and ab18058 (loading control to Vinculin) were both diluted 1/10,000 and incubated overnight at 4°C. 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 1/10,000 dilution for 1hr at room temperature before imaging.

BRG1 was immunoprecipitated from 0.35 mg HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10μg with Ab110641 at 1:20 dilution (0.3μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using Ab110641 1:1000 dilution (0.12 μg/ml). VeriBlot for IP Detection Reagent (HRP) (ab131366) was used as the secondary antibody at 1:1000 dilution.

Lane 1: HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate 10μg

Lane 2: Ab110641 IP in HeLa whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab110641 in HeLa whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 1 second 

Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling BRG1 with purified ab110641 at 1/200 dilution(10µg/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488)(1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control.

ab110641 staining BRG1 in wild-type HAP1 cells (top panel) and BRG1 knockout HAP1 cells (bottom panel). The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab110641 at 1/500 dilution and ab195889 at 1/250 dilution (shown in pseudocolour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

Immunohistochemical analysis of paraffin-embedded Rat liver tissue labeling BRG1 with ab110641, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Nuclear staining on rat liver. The section was incubated with ab229902 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0). Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Immunohistochemical analysis of paraffin-embedded Mouse kidney tissue labeling BRG1 with ab110641, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP). Nuclear staining on mouse kidney. The section was incubated with ab229902 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin. Heat mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

Immunohistochemical analysis of paraffin-embedded human testis tissue staining BRG1 with ab110641 at 1/100 dilution. Heat mediated antigen retrieval was perfomed with citrate buffer (pH 6).

Immunohistochemical analysis of paraffin-embedded human kidney tissue staining BRG1 with ab110641 at 1/100 dilution. Heat mediated antigen retrieval was perfomed with citrate buffer (pH 6).