- PA5-40421 - Invitrogen Antibodies KDM2A antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of human HepG2 cell lysate using an anti-FBXL11 polyclonal antibody (Product # PA5-40421).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of KDM2A was achieved by transfecting HeLa with KDM2A specific siRNAs (Silencer® select Product # s22790 and s22791). Western blot analysis (Fig. a) was performed using Nuclear enriched extracts from the KDM2A knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with KDM2A Polyclonal Antibody (Product # PA5-40421, 0.5 µg/mL) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:20,000 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 KDM2A. Uncharacterized band (*) was observed at around ~90 kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-KDM2A Polyclonal Antibody (Product # PA5-40421) and a 130 kDa band corresponding to KDM2A was observed across cell lines tested. Nuclear enriched extracts (30 µg lysate) of HeLa (Lane 1), T-47D (Lane 2), HCT 116 (Lane 3) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). 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 ( 0.5 µg/mL) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:20,000 dilution) using the iBright™ FL1500 Imaging System (Product # A44115). Chemiluminescent detection was performed using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Product # 34580).Uncharacterized bands (*) were observed around ~52 and ~90 kDa.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of KDM2A was performed using 70% confluent log phase HeLa 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 1 hour at room temperature. The cells were labeled with KDM2A Polyclonal Antibody (Product # PA5-40421) at 5 µg/mL in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A27034), (1:2000), 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). Panel d represents the merged image showing punctate nuclear 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 analysis of humanmuscle tissue using an anti-FBXL11 polyclonal antibody (Product # PA5-40421).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Chromatin Immunoprecipitation (ChIP) assay of endogenous KDM2A was performed using KDM2A Polyclonal Antibody (Product # PA5-40421, 5 µg) on sheared chromatin from MCF7 cells using the MAGnify ChIP System kit (Product # 49-2024). Normal Rabbit IgG was used as a negative IP control. The purified DNA was analyzed by qPCR using primers binding to CTNNA1 (doi:10.1080/19491034.2017.1342915), 3990-4092 region in Human rDNA, 12885-12970 region in Human rDNA (doi.org/10.1038/emboj.2010.56) (active) and SAT alpha (inactive). Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.

PA5-40421