Anti-hnRNP A2B1 antibody [EPR24002-81]

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-hnRNP A2B1 antibody [EPR24002-81] (AB259894)

IHC image of hnRNP A2B1 staining in a section of frozen normal human uterus performed on a Leica Biosystems BOND® RX instrument using the standard protocol. The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab259894, 0.05 μg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody. For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times. Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-hnRNP A2B1 antibody [EPR24002-81] (AB259894)

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

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-hnRNP A2B1 antibody [EPR24002-81] (AB259894)

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).

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-hnRNP A2B1 antibody [EPR24002-81] (AB259894)

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.

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-hnRNP A2B1 antibody [EPR24002-81] (AB259894)

IHC image of hnRNP A2B1 staining in a section of frozen normal human colon performed on a Leica Biosystems BOND® RX instrument using the standard protocol. The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab259894, 0.05 μg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody. For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-hnRNP A2B1 antibody [EPR24002-81] (AB259894)

IHC image of hnRNP A2B1 staining in a section of frozen normal mouse large intestine performed on a Leica Biosystems BOND® RX instrument using the standard protocol. The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab259894, 0.05 μg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody. For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• IHC-Fr

Lab

Immunohistochemistry (Frozen sections) - Anti-hnRNP A2B1 antibody [EPR24002-81] (AB259894)

IHC image of hnRNP A2B1 staining in a section of frozen normal mouse testis performed on a Leica Biosystems BOND® RX instrument using the standard protocol. The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab259894, 0.05 μg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody. For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-hnRNP A2B1 antibody [EPR24002-81] (AB259894)

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

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-hnRNP A2B1 antibody [EPR24002-81] (AB259894)

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.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-hnRNP A2B1 antibody [EPR24002-81] (AB259894)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-hnRNP A2B1 antibody [EPR24002-81] (AB259894)

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

• WB

Lab

Western blot - Anti-hnRNP A2B1 antibody [EPR24002-81] (AB259894)

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

Exposure time : 26 seconds

All lanes:

Western blot - Anti-hnRNP A2B1 antibody [EPR24002-81] (ab259894) at 1/1000 dilution

Lane 1:

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

Lane 2:

A431 (human epidermoid carcinoma epithelial cell), whole cell lysate at 20 µg

Lane 3:

MCF7 (human breast adenocarcinoma epithelial cell), whole cell lysate at 20 µg

Lane 4:

K562 (human chronic myelogenous leukemia lymphoblast), whole cell lysate at 20 µg

Lane 5:

HepG2 (human hepatocellar carcinoma epithelial cell), whole cell lysate at 20 µg

Lane 6:

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

Lane 7:

RAW264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage), whole cell lysate at 20 µg

Lane 8:

PC-12 (rat adrenal gland pheochromocytoma), whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 37 kDa

Observed band size: 36 kDa,38 kDa

false

• WB

Lab

Western blot - 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.

All lanes:

Western blot - Anti-hnRNP A2B1 antibody [EPR24002-81] (ab259894) at 1/1000 dilution

Lane 1:

Wild-type HEK-293T (human embryonic kidney epithelial cell), whole cell lysate at 20 µg

Lane 2:

hnRNP A2B1 knockout HEK-293T (human embryonic kidney epithelial cell), whole cell lysate at 20 µg

Lane 2:

Western blot - Human HNRNPA2B1 knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-hnrnpa2b1-knockout-hek-293t-cell-line-ab266404'>ab266404</a>)

Lane 3:

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

Lane 4:

A431 (human epidermoid carcinoma epithelial cell), whole cell lysate at 20 µg

Lane 5:

K562 (human chronic myelogenous leukemia lymphoblast), whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG H&L (IRDye® 800CW) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) and Goat Anti-Mouse IgG H&L (IRDye® 680RD) (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/10000 dilution

Predicted band size: 37 kDa

Observed band size: 36 kDa,38 kDa

false