- PA5-17361 - Invitrogen Antibodies KDM1A antibody

Ourailidou ME, Lenoci A, Zwergel C, Rotili D, Mai A, Dekker FJ
Bioorganic & medicinal chemistry 2017 Feb 1;25(3):847-856

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-LSD1 Polyclonal Antibody (Product # PA5-17361) and a 110 kDa band corresponding to LSD1 was observed across all tested cell lines. Nuclear enriched extracts (40 µg lysate) of K-562 (Lane 1), MCF7 (Lane 2), THP-1 (Lane 3), HeLa (Lane 4) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). 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 (1:1000 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # 27036,1:20000 dilution) using the iBright™ FL1500 Imaging System (Product # A44115). Chemiluminescent detection was performed using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Product # 34580).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of LSD1 was achieved by transfecting MCF7 with LSD1 specific siRNAs (Silencer® select Product # s617, s619). Western blot analysis (Fig. a) was performed using Nuclear enriched extracts from the LSD1 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with LSD1 Polyclonal Antibody (Product # PA5-17361, 1:1000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:20000 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 LSD1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of LSD1 in cell lysates from Hela and 293 cell lines using LSD1 polyclonal antibody (Product # PA5-17361).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of LSD1 was performed using 70% confluent log phase K-562 cells. The cells were fixed with 4% paraformaldehyde for 5 minutes, permeabilized with 0.1% Triton™ X-100 for 10 minutes, and blocked with 2% BSA for 1 hour at room temperature. The cells were labeled with LSD1 Polyclonal Antibody (Product # PA5-17361) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), (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). Panel d represents the merged image showing predominantly 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: Chromatin Immunoprecipitation (ChIP) assay of endogenous LSD1 protein using LSD1 Antibody: ChIP was performed using LSD1 Polyclonal Antibody (Product # PA5-17361, 5 µg) on sheared chromatin from A549 and A549 cells treated with Dexamethasone (100 nM, 2hours) 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 BIRC3, DUSP1 (active) and MYOD, SAT alpha (Inactive). Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.

PA5-17361