- MA5-24961 - Invitrogen Antibodies SATB1 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of SATB1 in HEK293T cells in untransfected (Left lane) and transfected (Right lane) samples using 5 µg per lane. The samples were separated by SDS-PAGE and probed with SATB1 (Product # MA5-24961) monoclonal antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of SATB1 was achieved by transfecting IMR-32 cells with SATB1 specific siRNAs (Silencer® select Product # s12481, Product # s12479). Western blot analysis (Fig. a) was performed using membrane extracts from the SATB1 knockdown cells (Lane 3), non-specific scrambled siRNA transfected cells (Lane 2) and untransfected cells (Lane 1). The blot was probed using Laminin gamma-1 Polyclonal Antibody (Product # MA5-24961, 1:2000 dilution) and Goat anti-Mouse IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to SATB1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-SATB1 Monoclonal Antibody (Product # MA5-24961) and a 100 kDa band corresponding to SATB1 was observed in cell line IMR-32 and Mouse thymus tissue. Whole cell extracts (30ug) of Jurkat (Lane 1), THP-1 (Lane 2), MCF-7 (Lane 3), MDA-MB-231 (Lane 4), IMR-32 (Lane 5), HEK-293 (Lane 6), tissue extracts (30ug) of Mouse Spleen (Lane 7), Rat Spleen (Lane 8), Mouse Thymus (Lane 9), Mouse Brain (Lane 10) and Mouse Kidney (Lane 11) 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 (1:2000 dilution) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L), Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). An uncharacterized band (*) corresponding to ~65 kDa and ~50 kDa was also observed in IMR-32 and in Mouse Thymus tissue.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of SATB1 in COS7 cells. Cells were transfected with a plasmid overexpressing SATB1 and probed with a SATB1 monoclonal antibody (Product # MA5-24961).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of SATB1 was performed using 70% confluent log phase HEK-293 cells. The cells were fixed with 4% Paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 10 minutes at room temperature. The cells were labeled with SATB1 Polyclonal Antibody (Product # MA5-24961) at 1:100 dilution in 0.1% BSA, incubated at 4 degree Celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody (Product # A27034) at a dilution of 1:2000 for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b: Blue) were stained with DAPI. F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300). Panel d represents the merged image showing Nuclear and Cytoplasmic localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry was performed on paraffin-embedded human kidney tissue. To expose target proteins, 10mM citric buffer, pH6.0, 100°C for 10min was used. Following antigen retrieval, tissues were probed with a SATB1 monoclonal antibody (Product # MA5-24961).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemistry was performed on paraffin-embedded human prostate tissue. To expose target proteins, 10mM citric buffer, pH6.0, 100°C for 10min was used. Following antigen retrieval, tissues were probed with a SATB1 monoclonal antibody (Product # MA5-24961).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Chromatin Immunoprecipitation (ChIP) assay of endogenous SATB1 protein using Anti-SATB1 Antibody: ChIP was performed using Anti-SATB1 Mouse Monoclonal Antibody (Product # MA5-24961, 5 µg) on sheared chromatin from IMR32 cells using the MAGnify ChIP System kit (Product # 49-2024). Normal Mouse IgG was used as a negative IP control. The purified DNA was analyzed by qPCR primers binding to ELAVL4 exonic region (+59kb), FOSB transcriptional start site, EGR2 intronic region (+2.5kb), LMNA exon 1 and SAT2 satellite repeats. Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.

MA5-24961