Recombinant Anti-hnRNP A2B1 antibody [EPR24002-81] (ab259894)

Blocking and diluting buffer and concentration: Intercept^® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS

Lanes 1- 4: Merged signal (red and green). Green - ab259894 observed at 36/38kDa. Red - loading control ab7291 (Mouse monoclonal [DM1A] to alpha Tubulin) observed at 50 kDa.

Lanes 1-2: ab259894 Anti-hnRNP A2B1 antibody was shown to react with hnRNP A2B1 in HEK-293T cells in Western blot. Loss of signal was observed when hnRNP A2B1 knockout cell line ab266404 (knockout cell lysate ab257224)  was used. Wild-type and hnRNP A2B1 knockout samples were subjected to SDS-PAGE.

ab259894 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated at 4°C overnight at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat Anti-Rabbit IgG H&L (IRDye^® 800CW) (ab216773) and Goat Anti-Mouse IgG H&L (IRDye^® 680RD) (ab216776) secondary antibodies at 1 in 10000 dilution for 1 hour at room temperature before imaging.

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

Exposure time: 26 seconds

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized 293T cells labelling hnRNP A2B1 with ab259894 at 1/1000 (0.525 ug/mL) dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150081) preadsorbed antibody at 1/1000 (2 ug/mL) dilution (Green). Confocal image showing nuclear staining in 293T cells and nearly no staining in HNRNPA2B1 knockout 293T cells. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5 ug/mL) dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150081) preadsorbed at 1/1000 (2 ug/mL) dilution.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 cells labelling hnRNP A2B1 with ab259894 at 1/1000 (0.525 ug/mL) dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150081) preadsorbed antibody at 1/1000 (2 ug/mL) dilution (Green). Confocal image showing nuclear staining in NIH/3T3 cells. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 (2.5 ug/mL) dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control: Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150081) preadsorbed at 1/1000 (2 ug/mL) dilution.

Immunohistochemical analysis of paraffin-embedded Human colon tissue labelling hnRNP A2B1 with ab259894 at 1/5000 (0.105 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on human colon. The section was incubated with ab259894 for 10 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 Citrate buffer (pH 6.0, epitope retrieval solution 1) for 10 mins

Immunohistochemical analysis of paraffin-embedded Mouse colon tissue labelling hnRNP A2B1 with ab259894 at 1/5000 (0.105 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on mouse colon. The section was incubated with ab259894 for 10 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 Citrate buffer (pH 6.0, epitope retrieval solution 1) for 10 mins

Immunohistochemical analysis of paraffin-embedded Rat colon tissue labelling hnRNP A2B1 with ab259894 at 1/5000 (0.105 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on rat colon. The section was incubated with ab259894 for 10 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 Citrate buffer (pH 6.0, epitope retrieval solution 1) for 10 mins

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Wild-type HEK-293T (human embryonic kidney epithelial cell) (Right) and hnRNP A2B1 knockout HEK-293T cells (Left) cells labelling hnRNP A2B1 with ab259894 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat F(ab')2 Anti-Rabbit IgG (DyLight® 488, ab98507) at 1/500 dilution was used as the secondary antibody. Positive staining on 293T cells (ab255449), while no staining on hnRNP A2B1 knockout HEK-293T cells (ab266404).

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 (Mouse embryonic fibroblast) cells labelling hnRNP A2B1 with ab259894 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat F(ab')2 Anti-Rabbit IgG(DyLight® 488, ab98507) at 1/500 dilution was used as the secondary antibody.