Recombinant Anti-BRD9 antibody [EPR23888-5] (ab259839)

All lanes : Anti-BRD9 antibody [EPR23888-5] (ab259839) at 1/1000 dilution

Lane 1 : Wild-type HEK-293T cell lysate
Lane 2 : BRD9 knockout HEK-293T cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Observed band size: 75 kDa why is the actual band size different from the predicted?

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 : Anti-BRD9 antibody [EPR23888-5] (ab259839) at 1/1000 dilution

Lane 1 : K-562 (human chronic myelogenous leukemia lymphoblast) whole cell lysate
Lane 2 : HDLM-2 (human hodgkin lymphoma ) whole cell lysate
Lane 3 : LNCaP (human prostate carcinoma epithelial cell) whole cell lysate
Lane 4 : Jurkat (human t cell leukemia t lymphocyte) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Observed band size: 80 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer and concentration: 5% NFDM/TBSTLysates used in this blot have experienced freeze-thaw cycles.

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

 

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

All lanes : Anti-BRD9 antibody [EPR23888-5] (ab259839) at 1/1000 dilution

Lanes 1 & 5 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : 293T (human embryonic kidney epithelial cell) whole cell lysate
Lane 3 : NIH/3T3 (mouse embryonic fibroblast) whole cell lysate
Lanes 4 & 6 : PC-12 (rat adrenal gland pheochromocytoma ) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Observed band size: 80 kDa why is the actual band size different from the predicted?

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.

 

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

All lanes : Anti-BRD9 antibody [EPR23888-5] (ab259839) at 1/1000 dilution

Lane 1 : Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate
Lane 2 : RAW 264.7 (mouse abelson murine leukemia virus-induced tumor macrophage) whole cell lysate
Lane 3 : C6 (rat glial tumor glial cell) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)

Observed band size: 80 kDa why is the actual band size different from the predicted?

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

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

 Exposure time: 3 minutes

 

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