Anti-hnRNP D/AUF1 antibody [EPR24001-12]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-hnRNP D/AUF1 antibody [EPR24001-12] (AB259895)

Immunohistochemical analysis of paraffin-embedded Human lung carcinoma tissue labelling hnRNP D/AUF1 with ab259895 at 1/100 (5.59 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on human lung carcinoma. The section was incubated with ab259895 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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-hnRNP D/AUF1 antibody [EPR24001-12] (AB259895)

Immunohistochemical analysis of paraffin-embedded Human breast tissue labelling hnRNP D/AUF1 with ab259895 at 1/100 (5.59 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on human breast. The section was incubated with ab259895 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

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-hnRNP D/AUF1 antibody [EPR24001-12] (AB259895)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa cells labelling hnRNP D/AUF1 with ab259895 at 1/50 (11.18 ug/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green) Confocal image showing nuclear staining in HeLa cells is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-hnRNP D/AUF1 antibody [EPR24001-12] (AB259895)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa (Human cervix adenocarcinoma cell) cells labelling hnRNP D/AUF1 with ab259895 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 anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

• IP

Supplier Data

Immunoprecipitation - Anti-hnRNP D/AUF1 antibody [EPR24001-12] (AB259895)

hnRNP D/AUF1 was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10 ug with ab259895 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab259895 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate 10 ug

Lane 2 : ab259895 IP in HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab259895 in HeLa whole cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 10 seconds

All lanes:

Immunoprecipitation - Anti-hnRNP D/AUF1 antibody [EPR24001-12] (ab259895)

Predicted band size: 38 kDa

Observed band size: 42 kDa

false

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-hnRNP D/AUF1 antibody [EPR24001-12] (AB259895)

Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labelling hnRNP D/AUF1 with ab259895 at 1/100 (5.59 ug/ml) dilution followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on mouse cerebrum. The section was incubated with ab259895 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

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-hnRNP D/AUF1 antibody [EPR24001-12] (AB259895)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 cells labelling hnRNP D/AUF1 with ab259895 at 1/50 (11.18 ug/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 dilution (Green) Confocal image showing nuclear staining in NIH/3T3 cells is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-hnRNP D/AUF1 antibody [EPR24001-12] (AB259895)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 (Mouse embryonic fibroblast) cells labelling hnRNP D/AUF1 with ab259895 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 anti rabbit IgG (Alexa Fluor® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

• IP

Supplier Data

Immunoprecipitation - Anti-hnRNP D/AUF1 antibody [EPR24001-12] (AB259895)

hnRNP D/AUF1 was immunoprecipitated from 0.35 mg NIH/3T3 (mouse embryonic fibroblast) whole cell lysate 10 ug with ab259895 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab259895 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : NIH/3T3 (mouse embryonic fibroblast) whole cell lysate 10 ug

Lane 2 : ab259895 IP in NIH/3T3 (mouse embryonic fibroblast) whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab259895 in NIH/3T3 whole cell lysate

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 3 minutes

All lanes:

Immunoprecipitation - Anti-hnRNP D/AUF1 antibody [EPR24001-12] (ab259895)

Predicted band size: 38 kDa

Observed band size: 42 kDa

false

• WB

Lab

Western blot - Anti-hnRNP D/AUF1 antibody [EPR24001-12] (AB259895)

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

Exposure time : 3 minutes

All lanes:

Western blot - Anti-hnRNP D/AUF1 antibody [EPR24001-12] (ab259895) at 1/1000 dilution

Lane 1:

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:

Jurkat (human T cell leukemia cell line from peripheral blood) whole cell lysate at 20 µg

Lane 4:

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

Lane 5:

U-2 OS (human bone osteosarcoma epithelial 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 1/20000 dilution

Predicted band size: 38 kDa

Observed band size: 37 kDa,42 kDa

false

• WB

Lab

Western blot - Anti-hnRNP D/AUF1 antibody [EPR24001-12] (AB259895)

False colour image of Western blot : Anti-hnRNP D/AUF1 antibody [EPR24001-12] staining at 1/1000 dilution, shown in green; loading control ab7291 (Mouse anti-Alpha Tubulin [DM1A]) staining at 1/20000 dilution, shown in red. In Western blot, ab259895 was shown to bind specifically to hnRNP D/AUF1. A band was observed at 40 kDa in wild-type U-2 OS cell lysates with no signal observed at this size in HNRNPD knockout cell line ab273860 (knockout cell lysate ab273814). To generate this image, wild-type and HNRNPD knockout U-2 OS cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % 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-hnRNP D/AUF1 antibody [EPR24001-12] (ab259895) at 1/1000 dilution

Lane 1:

Wild-type U-2 OS cell lysate at 20 µg

Lane 2:

HNRNPD knockout U-2 OS cell lysate at 20 µg

Lane 2:

Western blot - Human HNRNPD knockout U-2 OS cell line (<a href='/en-us/products/cell-lines/human-hnrnpd-knockout-u-2-os-cell-line-ab273860'>ab273860</a>)

Predicted band size: 38 kDa

Observed band size: 40 kDa

false

• WB

Lab

Western blot - Anti-hnRNP D/AUF1 antibody [EPR24001-12] (AB259895)

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

Exposure time : 81 seconds

All lanes:

Western blot - Anti-hnRNP D/AUF1 antibody [EPR24001-12] (ab259895) at 1/1000 dilution

Lane 1:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 40 µg

Lane 2:

C6 (rat glial tumor glial cell) whole cell lysate at 40 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 1/20000 dilution

Predicted band size: 38 kDa

Observed band size: 42 kDa

false