Anti-ADA 抗体 [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.

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

Exposure time: 

Lane1: 3.25 second;
Lane2: 37 seconds. 

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)

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. 

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. 

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. 

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. 

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. 

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.

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.

Exposure time: 10 seconds