- 701856 - Invitrogen Antibodies WNT2B antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on whole cell extracts (30 µg lysate) of MDA-MB-231 (Lane 1), Daudi (Lane 2), Ramos (Lane 3) and Mouse Kidney (Lane 4). The blots were probed with Anti-WNT2B/13 Recombinant Rabbit Monoclonal Antibody (Product # 701856, 1-2 µg/mL) and detected by chemiluminescence using Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.4 µg/mL, 1:2500 dilution). A 43 kDa and 35kDa bands corresponding to WNT2B/13 was observed. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 10% Bis-Tris gel (Product # NP0301BOX), XCell SureLock™ Electrophoresis System (Product # EI0002) and Novex® Sharp Pre-Stained Protein Standard (Product # LC5800). Resolved proteins were then transferred onto a nitrocellulose membrane with iBlot® Dry Blotting System (Product # IB21001). The membrane was probed with the relevant primary and secondary Antibody following blocking with 5% skimmed milk. Chemiluminescent detection was performed using Pierce™ ECL Western blotting Substrate (Product # 32106).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence was performed on methanol fixed MDA-MB-231 cells for detection of WNT2B/13 using Anti-WNT2B/13 Recombinant Rabbit Monoclonal Antibody (Product # 701856, 2 µg/mL), alpha-Tubulin Monoclonal Antibody (Product # 32-2500, 1 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000), Goat anti-Mouse IgG Secondary Antibody, Alexa Fluor® 594conjugate (Product # A-11032, 1:400) respectively. Panel a) shows representative cells that were stained for detection and localization of WNT2B/13 protein (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938,). Panel c) represents cytoskeletal alpha-tubulin staining (red). Panel d) is a composite image of Panels a, b and c clearly demonstrating cytoplasmic localization of WNT2B/13. Panel e) represents control cells with no primary antibody to assess background.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of Wnt 2B/13 showing staining in the cytoplasm and nucleus of paraffin-embedded human pancreas tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Wnt 2B/13 Recombinant Rabbit Monoclonal Antibody (Product # 701856) diluted in 3% BSA-PBS at a dilution of 1:20 for 1 hour at 37ºC in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunohistochemistry analysis of Wnt 2B/13 showing staining in the cytoplasm and nucleus of paraffin-embedded human kidney tissue (right) compared to a negative control without primary antibody (left). To expose target proteins, antigen retrieval was performed using 10mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with a Wnt 2B/13 Recombinant Rabbit Monoclonal Antibody (Product # 701856) diluted in 3% BSA-PBS at a dilution of 1:20 for 1 hour at 37ºC in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

701856