- PA5-50857 - Invitrogen Antibodies NSD1 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using NSD1 Polyclonal Antibody (Product # PA5-50857) and a 300 kDa band along with uncharacterized band (*) at 50 KDa corresponding to NSD1 was observed across cell lines tested. Fresh whole cell extracts (40 µg lysate) of HEK-293 (Lane 1), K-562 (Lane 2) were electrophoresed using NuPAGE™ 3-8% Tris-Acetate Protein Gel (Product # EA0378BOX). Resolved proteins was equilibrated with 20% ethanol and 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 # A27036, 1:20,000 dilution) using the iBright™ FL1500 Imaging System (Product # A44115). Chemiluminescent detection was performed using SuperSignal™ West Atto Ultimate Sensitivity Substrate (Product # A38556).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of NSD1 was achieved by transfecting HEK-293 with NSD1 specific siRNAs (Silencer® select Product # s34630, s34631). Western blot analysis (Fig. a) was performed using fresh whole cell extracts from the NSD1 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with NSD1 Polyclonal Antibody (Product # PA5-50857, 1:1000 dilution) 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 NSD1.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of NSD1 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 1 hour at room temperature. The cells were labeled with NSD1 Polyclonal Antibody (Product # PA5-50857) 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 dilution). Panel d represents the merged image showing 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: Immunofluorescence analysis of NSD1 was performed using 70% confluent log phase MCF7 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 NSD1 Polyclonal Antibody (Product # PA5-50857) 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 dilution). Panel d represents the merged image showing 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: Immunohistochemical analysis of NSD1 in paraffin embedded Human esophagus cancer tissue using NSD1 Polyclonal Antibody (Product # PA5-50857) at a 1:25 dilution.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Chromatin Immunoprecipitation (ChIP) was performed using NSD1 Polyclonal Antibody (Product # PA5-50857, 5 µg) on sheared chromatin from MCF7 cells using the MAGnify ChIP System (Product # 49-2024). Normal Rabbit IgG was used as a negative IP control. The purified DNA was analyzed by qPCR with PCR primer pairs over ZFP36L1, CTSD and KRT2 (active), and MYOD1 (inactive). We further analyzed the binding of NSD1 to different loci at BMP4 gene where Loci B region (-1655 bp) and C region (-1246 bp) bind to to NSD1 while D region (-683 bp) has lower binding affinity, while K region (+6161 bp) and L region (+7124 bp) are negative binders for NSD1 from transcription start site. Antibody specificity was demonstrated by detection of enrichment of the target protein at specific gene loci. Data is presented as fold enrichment of the antibody signal versus the Mouse Isotype using the comparative CT method.

PA5-50857