- 720030 - Invitrogen Antibodies SALL4 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 (20 µg lysate) of HeLa (Lane 1), Mouse Kidney (Lane 2), Ramos (Lane 3), K562 (Lane 4), Daudi (Lane 5), Raji (Lane 6), NIH/3T3 (Lane 7) and Cos7 (Lane 8). The blots were probed with Anti-Sall4 Rabbit Polyclonal antibody (Product # 720030, 1-3 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 clear 65 kDa band corresponding to Sall4 was observed across 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: Immunofluorescence was performed on fixed and permeabilized A-431cells for detection of Sall4 using Anti-Sall4 Rabbit Polyclonal Antibody (Product # 720030, 2 µg/mL) and labeled with Goat anti-Rabbit IgG (H+L) Superclonal Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034, 0.4 µg/mL, 1:2500). Panel a) shows representative cells that were stained for detection and localization of Sall4 protein (green), Panel b) is stained for nuclei (blue) using SlowFade® Gold Antifade Mountant with DAPI (Product # S36938, 1:50). Panel c) represents cytoskeletal F-actin staining using Alexa Fluor® 594 Phalloidin (Product # A12381, 1:200). Panel d) is a composite image of Panels a, b and c clearly demonstrating nuclear localization of Sall4. Panel e) represents control cells with no primary antibody to assess background.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of Sall4 (green) in Episomal Human iPSC cells. The cells were fixed with formalin for 15 minutes, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes, and blocked with 3% BSA in PBS (Product # 37525) for 30 minutes at room temperature. Cells were stained with a Sall4 polyclonal antibody (Product # 720030), at a dilution of 2 µg/mL for one hour at room temperature, and then incubated with a goat anti-rabbit Superclonal IgG secondary antibody, Alexa Fluor 488 conjugate (Product # A27034) at a dilution of 1:1000 for 30 minutes at room temperature (green). Nuclei (blue) were counterstained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific EVOS FL at 40X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of Sall4 (red) in human ESCs WIRB3 and HES2. The cells were fixed with 300 uL 4% paraformaldehyde solution for 15 min at RT in the dark, permeabilized with 300 uL 0.25% TritonX-100 solution (diluted in PBS) for 5 min at RT, and blocked with 300 uL 10% BSA solution (diluted in PBS) for 30 min in a 37C incubator. Cells were stained with a Sall4 polyclonal antibody (Product # 720030) at a dilution of 1:250 (2 µg/mL) in 3% BSA solution (diluted in PBS) at 4C overnight, and then incubated with a secondary antibody (Red). Nuclei (blue) was stained with DAPI (Product # D1306). Images were taken on a Leica DM5500 microscope at 10X magnification. Data courtesy of Dr. Jianlong Wang's lab.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry analysis of Sall4 showing staining in the nucleus of paraffin-embedded mouse testis 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 Sall4 Rabbit Polyclonal Antibody (Product # 720030) diluted in 3% BSA-PBS at a dilution of 1:100 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.

720030