Anti-BRG1 antibody [EPNCIR111A] - BSA and Azide free

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-BRG1 antibody [EPNCIR111A] - BSA and Azide free (AB215998)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab110641).

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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-BRG1 antibody [EPNCIR111A] - BSA and Azide free (AB215998)

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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab110641).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BRG1 antibody [EPNCIR111A] - BSA and Azide free (AB215998)

This IHC data was generated using the same anti-BRG1 antibody clone, EPNCIR111A, in a different buffer formulation (ab110641).
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).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-BRG1 antibody [EPNCIR111A] - BSA and Azide free (AB215998)

Clone EPNCIR111A (ab215998) has been successfully conjugated by Abcam. This image was generated using Anti-BRG1 antibody [EPNCIR111A] (Alexa Fluor® 647). Please refer to ab196535 for protocol details.

ab196535 staining BRG1 in SW480 cells. The cells were fixed with 100% methanol (5 min), 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 overnight at +4°C with ab196535 at a 1/100 dilution (shown in red) and ab195887, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 488), at a 1/250 dilution (shown in green). Nuclear DNA was labelled with DAPI (shown in blue).

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

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BRG1 antibody [EPNCIR111A] - BSA and Azide free (AB215998)

ab110641 at 1/100 dilution staining BRG1 in Human kidney tissue by Immunohistochemistry, Paraffin-embedded tissue.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab110641).

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

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-BRG1 antibody [EPNCIR111A] - BSA and Azide free (AB215998)

Clone EPNCIR111A (ab215998) has been successfully conjugated by Abcam. This image was generated using Anti-BRG1 antibody [EPNCIR111A] (Alexa Fluor® 488). Please refer to ab196314 for protocol details.

ab196314 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 overnight at +4°C with ab196314 at a 1/500 dilution (shown in green) and ab195889, Mouse monoclonal to alpha Tubulin (Alexa Fluor® 594), at a 1/250 dilution (shown in red). Nuclear DNA was labelled with DAPI (shown in blue).

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

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-BRG1 antibody [EPNCIR111A] - BSA and Azide free (AB215998)

Clone EPNCIR111A (ab215998) has been successfully conjugated by Abcam. This image was generated using Anti-BRG1 antibody [EPNCIR111A] (PE). Please refer to ab225124 for protocol details.

Overlay histogram showing HeLa cells stained with ab225124 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab225124, 1/500 dilution) for 30 min at 22°C.

Isotype control antibody (black line) was Rabbit IgG (monoclonal) Phycoerythrin (ab209478) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5,000 events were collected using a 50 mW Yellow/Green laser (561nm) and 586/15 bandpass filter.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-BRG1 antibody [EPNCIR111A] - BSA and Azide free (AB215998)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab110641).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-BRG1 antibody [EPNCIR111A] - BSA and Azide free (AB215998)

This ICC/IF data was generated using the same anti-BRG1 antibody clone, EPNCIR111A, in a different buffer formulation (cat# ab110641).

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).

• WB

Lab

Western blot - Anti-BRG1 antibody [EPNCIR111A] - BSA and Azide free (AB215998)

This data was developed using the same antibody clone in a different buffer formulation (ab110641).

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.

All lanes:

Western blot - Anti-BRG1 antibody [EPNCIR111A] (<a href='/en-us/products/primary-antibodies/brg1-antibody-epncir111a-ab110641'>ab110641</a>) at 1/10000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

SMARCA4 knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human SMARCA4 (BRG1) knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-smarca4-brg1-knockout-hek-293t-cell-line-ab255432'>ab255432</a>)

Predicted band size: 185 kDa

Observed band size: 185 kDa

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• WB

Lab

Western blot - Anti-BRG1 antibody [EPNCIR111A] - BSA and Azide free (AB215998)

This data was developed using ab110641, the same antibody clone in a different buffer formulation. Different batches of ab110641 were tested on HeLa (Human cervix adenocarcinoma epithelial cell) lysate at 0.5 µg/ml. 15 µg of lysate was loaded in each lane. Bands observed at 185 kDa.

All lanes:

Western blot - Anti-BRG1 antibody [EPNCIR111A] (<a href='/en-us/products/primary-antibodies/brg1-antibody-epncir111a-ab110641'>ab110641</a>)

Predicted band size: 185 kDa

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• WB

Lab

Western blot - Anti-BRG1 antibody [EPNCIR111A] - BSA and Azide free (AB215998)

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.

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

SMARCA4 knockout HEK-293T cell lysate at 20 µg

Predicted band size: 185 kDa

Observed band size: 185 kDa

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• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-BRG1 antibody [EPNCIR111A] - BSA and Azide free (AB215998)

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 found in the Protocol booklet in the Product Protocol section. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods. This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab110641).