Recombinant Anti-NSD3 antibody [EPR25189-8] (ab300489)

Blocking / Diluting buffer: Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS.

False colour image of Western blot: Anti-NSD3 antibody [EPR25189-8] (ab300489) staining at 1/2000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red.

In Western blot, ab300489 was shown to bind specifically to NSD3. Two bands were observed at 180kDa, 85kDa in wild-type HAP1 cell lysates with no signal observed at this size in the NSD3 knockout cell line. To generate this image, wild-type and NSD3 knockout HAP1 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a PVDF-FL membrane. Membranes were blocked in Odyssey diluted in equal volume of 0.1 % TBS 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.

Performed under reducing conditions.

Blocking / Diluting buffer: 5% NFDM/TBST.

The expression profile and molecular weight observed are consistent with what has been described in the literature (PMID: 34615858, 26626481).

This blot was developed using a high sensitivity ECL substrate.

Lysates were freshly made and used immediately to minimize protein degradation.

Immunohistochemical analysis of paraffin-embedded cell pellets.
Panel A: Wild-type HAP1 (Human chronic myelogenous leukemia near-haploid cell) cell pellet.
Panel B: NSD3 knockout HAP1 cell pellet.
NSD3 labeled with ab300489 at 1/500 dilution (0.998 µg/ml) followed by a ready to use Leica DS9800 (BOND™ Polymer Refine Detection) kit. Staining on (A) wild-type HAP1 cell pellet, no staining on (B) NSD3 knockout HAP1 cell pellet. The section was incubated with ab300489 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 LeicaDS9800 (BOND™ Polymer Refine Detection).

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

Immunohistochemical analysis of paraffin-embedded human kidney tissue labeling NSD3 with ab300489 at 1/500 dilution (0.998 µg/ml) followed by a ready to use Leica DS9800 (BOND™ Polymer Refine Detection) kit. Nuclear staining on human kidney. The section was incubated with ab300489 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 LeicaDS9800 (BOND™ Polymer Refine Detection).

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

Immunohistochemical analysis of paraffin-embedded mouse cardiac muscle tissue labeling NSD3 with ab300489 at 1/500 dilution (0.998 µg/ml) followed by a ready to use Leica DS9800 (BOND™ Polymer Refine Detection) kit. Nuclear staining on mouse cardiac muscle. The section was incubated with ab300489 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 LeicaDS9800 (BOND™ Polymer Refine Detection).

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

Immunohistochemical analysis of paraffin-embedded rat cardiac muscle tissue labeling NSD3 with ab300489 at 1/500 dilution (0.998 µg/ml) followed by a ready to use Leica DS9800 (BOND™ Polymer Refine Detection) kit. Nuclear staining on rat cardiac muscle. The section was incubated with ab300489 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 LeicaDS9800 (BOND™ Polymer Refine Detection).

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

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized wild type HAP1 and NSD3 KO HAP1 (NSD3 knockout human chronic myelogenous leukemia near-haploid cells) labeling NSD3 with ab300489 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150081) secondary antibody at 1/1000 dilution (green).

Confocal image showing nuclear staining in wild-type HAP1 cell line and no staining in NSD3 knockout HAP1 cell line. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used as a counterstain (red). The nuclear counter stain used is DAPI (blue).

The lower panels show secondary only controls where secondary but no primary antibody was added. The nuclear counter stain used is DAPI (blue).

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (mouse embryonic fibroblasts) labeling NSD3 with ab300489 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150081) secondary antibody at 1/1000 dilution (green).

Confocal image showing nuclear staining in NIH/3T3 cell line. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used as a counterstain (red). The nuclear counter stain used is DAPI (blue).

Flow Cytometry (Intracellular) analysis of Wild-type HAP1 (human chronic myelogenous leukemia near-haploid cell, Right) / NSD3 knockout HAP1(Left) labeling NSD3 (red) with ab300489 at a 1/500 dilution. Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. A Goat Anti-Rabbit IgG (Alexa Fluor^® 488, (ab150081)) was used as the secondary antibody at a 1/2000 dilution. Black - Rabbit monoclonal IgG (ab172730) Isotype control. Blue - (unlabeled control) - Cells without incubation with primary and secondary antibodies. 

Flow Cytometry (Intracellular) analysis of NIH/3T3 (mouse embryonic fibroblasts) labeling NSD3 (red) with ab300489 at a 1/500 dilution. Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol. A Goat Anti-Rabbit IgG (Alexa Fluor^® 488, (ab150081)) was used as the secondary antibody at a 1/2000 dilution. Black - Rabbit monoclonal IgG (ab172730) Isotype control. Blue - (unlabeled control) - Cells without incubation with primary and secondary antibodies.

SD3 was immunoprecipitated from 10 μg HAP1 (human chronic myelogenous leukemia near-haploid cell line) whole cell lysate with ab300489 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab300489 at 1/1000 dilution. VeriBlot for IP secondary antibody (HRP) (ab131366), was used as secondary antibody at 1/5000 dilution.

Lane 1: HAP1 whole cell lysate 10 μg (Input).

Lane 2: HAP1 whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab300489 in HAP1 whole cell lysate.

Blocking and dilution buffer: 5% NFDM/TBST.
Exposure time: 125 seconds.

Observed MW(KDa): 180,85

This blot was developed using a high sensitivity ECL substrate.
Lysates were freshly made and used immediately to minimize protein degradation.
Extra bands at 70 and 50 kDa observed in lane 2 may be caused by degradation during incubation.