- 711048 - Invitrogen Antibodies SOX9 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 Caco-2 (Lane 1), Hep G2 (Lane 2), PC-3 (Lane 3) and HEK-293 (Lane 4). The blots were probed with Anti-Sox9 Recombinant Rabbit Polyclonal Antibody (Product # 711048, 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 70 kDa band corresponding to Sox9 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 analysis of SOX9 was performed by loading 30 µg of PC3 wild type (Lane 1), PC3 Cas9 control (Lane 2), PC3 SOX9 knockout (Lane 3) whole cell extracts. The blot was probed with Anti-SOX9 Polyclonal Antibody (Product # 711048) (1 µg/mL) and Goat anti-Rabbit IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036) (1:4000 dilution). Loss of signal upon CRISPR mediated knockout (KO) confirms that antibody is specific to SOX9.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-SOX9 Polyclonal Antibody, (Product # 711048) and a ~70 kDa band corresponding to post translationally modified SOX2 was observed across the panel tested except THP-1 and Daudi which are reported to be negative. Another band at ~54 kDa (*) corresponding to unmodified SOX9 was also observed across the SOX9 positive cell lines. An uncharacterized band (**) at ~60kDa was observed in PC-3. Modified whole cell extracts (1% SDS) (30 µg lysate) of U-2 OS (Lane 1), PANC-1 (Lane 2), PC-3 (Lane 3), HEK-293 (Lane 4), THP-1 (Lane 5) and Daudi (Lane 6) were electrophoresed using Novex® NuPAGE® 4-12% % Bis-Tris gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (2µg/ml) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005)..

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: For immunofluorescence analysis, HCT 116 cells were fixed and permeabilized for detection of endogenous SOX9 using Anti- SOX9 Recombinant Rabbit Polyclonal Antibody (Product # 711048, 2 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 1:2000). Panel a) shows representative cells that were stained for detection and localization of SOX9 protein (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938). Panel c) represents cytoskeletal F-actin staining using Alexa Fluor® 555 Rhodamine Phalloidin (Product # R415, 1:300). Panel d) is a composite image of Panels a, b and c clearly demonstrating nuclear localization of SOX9. Panel e) represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

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