Anti-BRD9 antibody [EPR23888-5] - BSA and Azide free

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BRD9 antibody [EPR23888-5] - BSA and Azide free (AB277488)

This data was developed using ab259839, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human testis tissue labelling BRD9 with ab259839 at 1/500 dilution (0.908 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear and weak cytoplasmic staining on human testis. The section was incubated with ab259839 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BRD9 antibody [EPR23888-5] - BSA and Azide free (AB277488)

This data was developed using ab259839, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Mouse testis tissue labelling BRD9 with ab259839 at 1/500 dilution (0.908 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on mouse testis. The section was incubated with ab259839 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-BRD9 antibody [EPR23888-5] - BSA and Azide free (AB277488)

This data was developed using ab259839, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Rat testis tissue labelling BRD9 with ab259839 at 1/500 dilution (0.908 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear and weak cytoplasmic staining on rat testis. The section was incubated with ab259839 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• WB

Lab

Western blot - Anti-BRD9 antibody [EPR23888-5] - BSA and Azide free (AB277488)

This data was developed using ab259839, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concentration : 5% NFDM/TBST

Lysates used in this blot have experienced freeze-thaw cycles.

Exposure time : Lanes 1-2 : 37 seconds;Lanes 3-4 : 3 minutes.

All lanes:

Western blot - Anti-BRD9 antibody [EPR23888-5] (<a href='/en-us/products/primary-antibodies/brd9-antibody-epr23888-5-ab259839'>ab259839</a>) at 1/1000 dilution

Lane 1:

K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate at 20 µg

Lane 2:

HDLM-2 (human hodgkin lymphoma ) whole cell lysate at 20 µg

Lane 3:

LNCaP (human prostate carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 4:

Jurkat (human t cell leukemia t lymphocyte) whole cell lysate 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 1/100000 dilution

Predicted band size: 67 kDa

Observed band size: 80 kDa

false

• WB

Lab

Western blot - Anti-BRD9 antibody [EPR23888-5] - BSA and Azide free (AB277488)

False colour image of Western blot : Anti-BRD9 antibody [EPR23888-5] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab259839 was shown to bind specifically to BRD9. A band was observed at 75 kDa in wild-type HEK-293T cell lysates with no signal observed at this size in BRD9 knockout cell line ab266763 (knockout cell lysate ab258336). To generate this image, wild-type and BRD9 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 5 % 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.

All lanes:

Western blot - Anti-BRD9 antibody [EPR23888-5] (<a href='/en-us/products/primary-antibodies/brd9-antibody-epr23888-5-ab259839'>ab259839</a>) at 1/1000 dilution

Lane 1:

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

Lane 2:

BRD9 knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human BRD9 knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-brd9-knockout-hek-293t-cell-line-ab266763'>ab266763</a>)

Predicted band size: 67 kDa

Observed band size: 75 kDa

false

• WB

Lab

Western blot - Anti-BRD9 antibody [EPR23888-5] - BSA and Azide free (AB277488)

This data was developed using ab259839, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concentration : 5% NFDM/TBST

The molecular weight observed is consistent with what has been described in the literature (PMID : 32457312; 31015438 ).

Lysates were made freshly and used in WB immediately to minimize protein degradation (lane1-4). Lanes 5-6 are the lysates from same cell lines but have experienced freeze-thaw cycles.

Exposure time : Lanes 1-4 : 37 seconds; Lanes 5-6 : 3 minutes.

All lanes:

Western blot - Anti-BRD9 antibody [EPR23888-5] (<a href='/en-us/products/primary-antibodies/brd9-antibody-epr23888-5-ab259839'>ab259839</a>) at 1/1000 dilution

Lanes 1 and 5:

HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 3:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lanes 4 and 6:

PC-12 (rat adrenal gland pheochromocytoma ) whole cell lysate 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 1/100000 dilution

Predicted band size: 67 kDa

Observed band size: 80 kDa

false

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-BRD9 antibody [EPR23888-5] - BSA and Azide free (AB277488)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab259839).

CUT&RUN profiling with BRD9 antibody demonstrates robust genome-wide enrichment in cells. Heatmaps of genome-wide signal flanking annotated transcription start sites (TSSs, +/- 2 kbp) display CUT&RUN data generated using the CUTANA™ CUT&RUN Kit (EpiCypher 14-1048) with BRD9 antibody (Abcam ab259839, 0.5 µg). 500,000 HEK293T cells were used per reaction. IgG antibody was included as a negative control to assess non-specific background. Libraries were prepared using the CUTANA™ CUT&RUN Library Prep Kit (EpiCypher 14-1001). Sequencing was performed with paired-end 50 bp reads, and data were processed on CUTANA™ Cloud (cloud.epicypher.com) by alignment to the hg38 genome. Heatmaps were generated using deepTools (Ramнrez et al., Nucleic Acids Res. 2014; PMID : 24799436). Row-linked data are ranked by intensity relative to BRD9 WT, with red indicating high localized enrichment and blue denoting background.

• ChIC/CUT&RUN-seq

Supplier Data

ChIC/CUT&RUN sequencing - Anti-BRD9 antibody [EPR23888-5] - BSA and Azide free (AB277488)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab259839).

CUT&RUN profiling with BRD9 antibody reveals the expected genomic enrichment pattern cells. Representative genome browser tracks show CUT&RUN data generated using the CUTANA™ CUT&RUN Kit (EpiCypher 14-1048) with BRD9 antibody (Abcam ab259839, 0.5 µg). 500,000 HEK293T cells were used per reaction. IgG, H3K4me3, and H3K27me3 antibodies were included as controls to assess non-specific background, active promoters, and repressed chromatin, respectively. Libraries were prepared using the CUTANA™ CUT&RUN Library Prep Kit (EpiCypher 14-1001). Sequencing was performed with paired-end 50 bp reads, and data were processed on CUTANA™ Cloud (cloud.epicypher.com) by alignment to the hg38 genome. Images were generated using Integrative Genomics Viewer (IGV, Broad Institute).

• WB

Lab

Western blot - Anti-BRD9 antibody [EPR23888-5] - BSA and Azide free (AB277488)

This data was developed using ab259839, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concentration : 5% NFDM/TBST

Lysates used in this blot have experienced freeze-thaw cycles.

Exposure time : 3 minutes

All lanes:

Western blot - Anti-BRD9 antibody [EPR23888-5] (<a href='/en-us/products/primary-antibodies/brd9-antibody-epr23888-5-ab259839'>ab259839</a>) at 1/1000 dilution

Lane 1:

Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate at 20 µg

Lane 2:

RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 3:

C6 (rat glial tumor glial cell) whole cell lysate 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 1/100000 dilution

Predicted band size: 67 kDa

Observed band size: 80 kDa

false