Anti-ADA antibody [EPR25429-117]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ADA antibody [EPR25429-117] (AB300050)

Immunohistochemical analysis of paraffin-embedded human cerebrum tissue labeling ADA with ab300050 at 1/2000 followed by a ready to use LeicaDS9800 (BOND^™ Polymer Refine Detection) was used. Negative control : no staining on human cerebrum (PMID : 2606352). The section was incubated with ab300050 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : PBS was used instead of primary antibody (ab300050) followed by ready to use secondary antibody 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-ADA antibody [EPR25429-117] (AB300050)

Immunohistochemical analysis of paraffin-embedded human tonsil tissue labelling ADA with ab300050 at 1/2000 followed by a ready to use LeicaDS9800 (BOND^™ Polymer Refine Detection). Positive staining on human tonsil (PMID : 30778076) (PMID : 6360330). The section was incubated with ab300050 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : PBS was used instead of primary antibody (ab300050) followed by ready to use secondary antibody 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-ADA antibody [EPR25429-117] (AB300050)

Immunohistochemical analysis of paraffin-embedded human thymus tissue labelling ADA with ab300050 at 1/2000 followed by a ready to use LeicaDS9800 (BOND^™ Polymer Refine Detection). Positive staining on human thymus (PMID : 2606352). The section was incubated with ab300050 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : PBS was used instead of primary antibody (ab300050) followed by ready to use secondary antibody 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-ADA antibody [EPR25429-117] (AB300050)

Immunohistochemical analysis of paraffin-embedded human stomach tissue labeling ADA with ab300050 at 1/2000 followed by a ready to use LeicaDS9800 (BOND^™ Polymer Refine Detection) was used. Positive staining on human stomach. The section was incubated with ab300050 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : PBS was used instead of primary antibody (ab300050) followed by ready to use secondary antibody 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-ADA antibody [EPR25429-117] (AB300050)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeilized Jurkat (human T cell leukemia T lymphocyte) cells lebelling ADA with ab300050 at 1/50 dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic and membranous staining in the Jurkat cell line.

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 : PBS was used instead of primary antibody followed by secondary antibody (ab150081) Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed at 1/1000 dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ADA antibody [EPR25429-117] (AB300050)

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling ADA with ab300050 at 1/2000 followed by a ready to use LeicaDS9800 (BOND^™ Polymer Refine Detection). Positive staining on the stromal cells of human liver. The section was incubated with ab300050 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control : PBS was used instead of primary antibody (ab300050) followed by ready to use secondary antibody LeicaDS9800 (BOND^™ Polymer Refine Detection).

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

• IP

Supplier Data

Immunoprecipitation - Anti-ADA antibody [EPR25429-117] (AB300050)

ADA was immunoprecipitated from 0.35 mg Jurkat (human T cell leukemia T lymphocyte) whole cell lysate 10 µg with ab300050 at 1/30 dilution. Western blot was performed on the immunoprecipitate using ab300050 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : Jurkat (human T cell leukemia T lymphocyte) whole cell lysate 10 µg (Input)

Lane 2 : ab300050 IP in Jurkat whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab300050 in Jurkat whole cell lysate

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

All lanes:

Immunoprecipitation - Anti-ADA antibody [EPR25429-117] (ab300050) at 1/1000 dilution

Lane 1:

Jurkat (human T cell leukemia T lymphocyte) whole cell lysate (Input) at 10 µg

Lane 2:

ab300050 IP in Jurkat whole cell lysate

Lane 3:

Rabbit monoclonal IgG (<a href='/en-us/products/primary-antibodies/rabbit-igg-monoclonal-epr25a-isotype-control-ab172730'>ab172730</a>) instead of ab300050 in Jurkat whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Predicted band size: 40 kDa

false

Exposure time: 10s

• WB

Supplier Data

Western blot - Anti-ADA antibody [EPR25429-117] (AB300050)

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

Exposure time :

Lane1 : 3.25 second;

Lane2 : 37 seconds.

All lanes:

Western blot - Anti-ADA antibody [EPR25429-117] (ab300050) at 1/1000 dilution

Lane 1:

Human thymus tissue lysate at 20 µg

Lane 2:

Human tonsil tissue lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 40 kDa

Observed band size: 41 kDa

false

• WB

Lab

Western blot - Anti-ADA antibody [EPR25429-117] (AB300050)

Western blot : Anti-Ada antibody [EPR25429-117] (ab300050) staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab300050 was shown to bind specifically to Ada. A band was observed at 41 kDa in wild-type A549 cell lysates with no signal observed at this size in Ada knockout cell line. To generate this image, wild-type and Ada knockout A549 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 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-ADA antibody [EPR25429-117] (ab300050) at 1/1000 dilution

Lane 1:

Wild-type A549 cell lysate at 20 µg

Lane 2:

Ada knockout A549 cell lysate at 20 µg

Lane 3:

Jurkat cell lysate at 20 µg

Lane 4:

A549 Nuclear cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 41 kDa

false

• WB

Supplier Data

Western blot - Anti-ADA antibody [EPR25429-117] (AB300050)

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

Exposure time :

Lane1-2 : 1 second;

Lane3-4 : 3.25 seconds.

Low expression : human liver(PMID : 2606352)

All lanes:

Western blot - Anti-ADA antibody [EPR25429-117] (ab300050) at 1/1000 dilution

Lane 1:

Jurkat (human T cell leukemia T lymphocyte) whole cell lysate at 20 µg

Lane 2:

MOLT-4 (human lymphoblastic leukemia T lymphoblast) whole cell lysate at 20 µg

Lane 3:

Human stomach tissue lysate at 20 µg

Lane 4:

Human liver tissue lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 40 kDa

Observed band size: 41 kDa,36 kDa

false