PA5-50857

antibody from Invitrogen Antibodies

Targeting: NSD1 ARA267, FLJ22263, KMT3B, STO

Western blot (Data presented on provider website)

Immunocytochemistry (Supportive data in Antibodypedia)

Immunohistochemistry (Data presented on provider website)

Chromatin Immunoprecipitation (Supportive data in Antibodypedia)

Antibody Data

Product number

PA5-50857

Provider

Invitrogen Antibodies

Product name

NSD1 Polyclonal Antibody

Antibody type

Polyclonal

Antigen

Synthetic peptide

Description

The antibody detects endogenous levels of total NSD1 / KMT3B protein.

Reactivity

Human

Host

Rabbit

Isotype

IgG

Vial size

100 µL

Concentration

1.5 mg/mL

Storage

-20°C

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

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)

Main image

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.

Chromatin Immunoprecipitation

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

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.