重组Anti-BRG1抗体[EPNCIR111A] (ab110641)

Immunohistochemical analysis of formalin fixed paraffin embedded human tonsil labelling BRG1 with ab110641 at a concentration of 0.1 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with an OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was conducted for 32 mins at 100°C with ULTRA cell conditioning solution (CC1, pH 8.5). ab110641 anti-BRG1 antibody [EPNCIR111A] was incubated at 37°C for 16 mins. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Immunohistochemical analysis of formalin fixed paraffin embedded human tonsil labelling BRG1 with ab110641 at a concentration of 0.1 µg/ml. The immunostaining was performed on a Leica Biosystems BOND® RX instrument with a Bond™ Polymer Refine Detection kit. Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution 2) for 20 mins. ab110641 anti-BRG1 antibody [EPNCIR111A] was incubated for 30 mins at room temperature. Sections were counterstained with Hematoxylin. Image inset shows absence of staining in secondary antibody only control.

ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL, 2.5 x 10^5 HeLa (Human cervix adenocarcinoma epithelial cell line) cells and 5µg of ab110641 [EPNCIR111A]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads. The negative IgG control ab172730 is also shown.

Additional screenshots of mapped reads can be downloaded here.

The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

Flow cytometry overlay histogram showing left wild-type Hap1 positive cells and right negative SMARCA4 knockout Hap1 stained with ab95363 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab110641) (1x 10⁶ in 100μl at 0.008 μg/ml (1/266250)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C

Isotype control antibody Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (black line) was used at the same concentration and conditions as the primary antibody. Unlabelled sample was also used as a control (blue line).

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

False colour image of Western blot: Anti-BRG1 antibody [EPNCIR111A] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab110641 was shown to bind specifically to BRG1. A band was observed at 185 kDa in wild-type HEK-293T cell lysates with no signal observed at this size in SMARCA4 knockout cell line ab255432 (knockout cell lysate ab263853). To generate this image, wild-type and SMARCA4 knockout HEK-293T cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

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. 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).

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

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

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 

Intracellular 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 Fluorr^® 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. 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. 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).