- 702374 - Invitrogen Antibodies CTNNB1 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 NTERA-2 (Lane 1), SW480 (Lane 2), HEK-293 (Lane 3), A-431 (Lane 4) and Caco-2 (Lane 5). The blots were probed with Anti-β-Catenin (pS552) Recombinant Rabbit Monoclonal Antibody (Product # 702374, 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 85 kDa band corresponding to β-Catenin (pS552) was observed across the cell lines tested. Known quantity of protein samples were electrophoresed using Novex® NuPAGE® 4-12% Bis-Tris gel (Product # NP0321BOX), 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)
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Experimental details: Western blot was performed using Anti-Phospho-beta Catenin (Ser552) Recombinant Rabbit Monoclonal Antibody (1H29L24) (Product # 702374) and a 85 kDa band corresponding to Catenin beta-1 was observed across the cell lines tested. Whole cell extracts (30 µg lysate) of A-431 (Lane 1), A-431 treated with 100 ng/mL of Human recombinant EGF for 6 hours (Lane 2), A-431 treated with 100 ng/mL of Human recombinant EGF for 6 hours and 25 µM of MG132 for 4 hours(Lane 3), MCF7 (Lane 4), MCF7 treated with 100 ng/mL of Human recombinant EGF for 6 hours(Lane 5 and MCF7 treated with 100 ng/mL of Human recombinant EGF for 6 hours and 25 µM of MG132 for 4 hours (Lane 6) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23002) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (at a concentration of 1 µg/mL) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036,1:20000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescentdetection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Immunofluorescence analysis of Catenin beta-1 was performed using 70% confluent log phase MCF7 cells treated with 100 ng/mL of Human Recombinant EGF for 6 hours. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with Phospho-beta Catenin (Ser552) Recombinant Rabbit Monoclonal Antibody (1H29L24) (Product # 702374) at a concentration of 1 µg/mL in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32731), (1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b:Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300 dilution). Panel d represents the merged image showing nuclear translocation of the protein. Panel e represents untreated MCF7 cells showing no expression. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

702374