Anti-BRG1 antibody [EPR3912]

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BRG1 antibody [EPR3912] (AB108318)

Immunohistochemical analysis of formalin fixed paraffin embedded human tonsil labelling BRG1 with ab108318 at a concentration of 4 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

ab108318 Anti-BRG1 antibody [EPR3912] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual)

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-BRG1 antibody [EPR3912] (AB108318)

ab108318 (unpurified) 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 ab108318 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).

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-BRG1 antibody [EPR3912] (AB108318)

Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling BRG1 with purified ab108318 at 1/20 dilution (10μg/ml) (red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluorr^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-BRG1 antibody [EPR3912] (AB108318)

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling BRG1 with purified ab108318 at 1/50 dilution (2.3 μg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at 1/200 (2.5 μg/ml). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 μg/ml) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-BRG1 antibody [EPR3912] (AB108318)

ab108318 (unpurified) 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 ab108318 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).

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BRG1 antibody [EPR3912] (AB108318)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human bladder cancer tissue sections labeling BRG1 with purified ab108318 at 1/500 dilution (0.23 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• IP

Lab

Immunoprecipitation - Anti-BRG1 antibody [EPR3912] (AB108318)

BRG1 was immunoprecipitated from K-562 (human chronic myelogenous leukemia lymphoblast), whole cell lysate with ab108318 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab108318 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used as secondary antibody at 1/5000 dilution. Lane 1 (Input) : K-562 (human chronic myelogenous leukemia lymphoblast), whole cell lysate, 10 μg Lane 2 (+) : K-562 whole cell lysate Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab108318 in K-562 whole cell lysate Blocking and diluting buffer and concentration : 5% NFDM/TBST. Observed MW : 185 kDa.

All lanes:

Immunoprecipitation - Anti-BRG1 antibody [EPR3912] (ab108318) at 1/1000 dilution

Lane 1:

K-562 (human chronic myelogenous leukemia lymphoblast), whole cell lysate at 10 µg

Lane 2:

K-562 whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Predicted band size: 185 kDa

Observed band size: 185 kDa

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• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BRG1 antibody [EPR3912] (AB108318)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Mouse stomach tissue sections labeling BRG1 with purified ab108318 at 1/500 dilution (0.23 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BRG1 antibody [EPR3912] (AB108318)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Rat stomach tissue sections labeling BRG1 with purified ab108318 at 1/500 dilution (0.23 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control : PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

• IP

Lab

Immunoprecipitation - Anti-BRG1 antibody [EPR3912] (AB108318)

BRG1 was immunoprecipitated from NIH/3T3 (mouse embryonic fibroblast), whole cell lysate with ab108318 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab108318 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used as secondary antibody at 1/5000 dilution. Lane 1 (Input) : NIH/3T3 (mouse embryonic fibroblast), whole cell lysate, 10 μg Lane 2 (+) : NIH/3T3 whole cell lysate Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab108318 in NIH/3T3 whole cell lysate Blocking and diluting buffer and concentration : 5% NFDM/TBST. Observed MW : 185 kDa.

All lanes:

Immunoprecipitation - Anti-BRG1 antibody [EPR3912] (ab108318) at 1/1000 dilution

Lane 1:

NIH/3T3 (mouse embryonic fibroblast), whole cell lysate at 10 µg

Lane 2:

NIH/3T3 whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Predicted band size: 185 kDa

Observed band size: 185 kDa

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• WB

Lab

Western blot - Anti-BRG1 antibody [EPR3912] (AB108318)

All lanes:

Western blot - Anti-BRG1 antibody [EPR3912] (ab108318) at 1/1000 dilution

Lane 1:

K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysates at 20 µg

Lane 2:

NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates at 20 µg

Lane 3:

PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysates at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 185 kDa

Observed band size: 185 kDa

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• WB

Lab

Western blot - Anti-BRG1 antibody [EPR3912] (AB108318)

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

ab108318 was shown to specifically react with BRG1 in wild-type HAP1 cells along with additional cross reactive bands. No band was observed when BRG1 knockout samples were used. Wild-type and BRG1 knockout samples were subjected to SDS-PAGE, ab108318 and ab18058 (loading control to Vinculin) were diluted 1/1000 and 1/10,000 respectively 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.

All lanes:

Western blot - Anti-BRG1 antibody [EPR3912] (ab108318) at 1/1000 dilution

Lane 1:

Wild-type HAP1 cell lysate at 20 µg

Lane 2:

BRG1 knockout HAP1 cell lysate at 20 µg

Lane 3:

K562 cell lysate at 20 µg

Lane 4:

Molt-4 cell lysate at 20 µg

Predicted band size: 185 kDa

Observed band size: 185 kDa

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• WB

Lab

Western blot - Anti-BRG1 antibody [EPR3912] (AB108318)

Different batches of ab108318 were tested on K-562 (Human chronic myelogenous leukemia lymphoblast) lysate at 0.05 µg/ml. 15 µg of lysate was loaded in each lane. Bands observed at 185 kDa.

All lanes:

Western blot - Anti-BRG1 antibody [EPR3912] (ab108318)

Predicted band size: 185 kDa

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• WB

Lab

Western blot - Anti-BRG1 antibody [EPR3912] (AB108318)

Lanes 1- 2 : Merged signal (red and green). Green - ab108318 observed at 185 kDa. Red - Anti-Vinculin antibody [VIN-54] observed at 124 kDa.

ab108318 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. ab108318 and Anti-Vinculin antibody [VIN-54] 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 [EPR3912] (ab108318) 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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• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-BRG1 antibody [EPR3912] (AB108318)

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 ab108318 [EPR3912]. 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.

• WB

Lab

Western blot - Anti-BRG1 antibody [EPR3912] (AB108318)

All lanes:

Western blot - Anti-BRG1 antibody [EPR3912] (ab108318) at 1/10000 dilution

All lanes:

RAW264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysates at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 185 kDa

Observed band size: 185 kDa

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• WB

Unknown

Western blot - Anti-BRG1 antibody [EPR3912] (AB108318)

All lanes:

Western blot - Anti-BRG1 antibody [EPR3912] (ab108318) at 1/1000 dilution

Lane 1:

K562 cell lysate at 10 µg

Lane 2:

Molt-4 cell lysate at 10 µg

Predicted band size: 185 kDa

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