- PA5-30581 - Invitrogen Antibodies DNMT1 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of DNMT1 using A) 30 µg Jurkat whole cell extract (B) 30 µg Raji whole cell extract and C) 30 µg NCI-H929 whole cell extract. Samples were loaded onto a 5% SDS-PAGE gel and probed with a DNMT1 polyclonal antibody (Product # PA5-30581) at a dilution of 1:500.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of DNMT1 using A) 30 µg 293T whole cell extract (B) 30 µg A431 whole cell extract (C) 30 µg HeLa whole cell extract and D) 30 µg HepG2 whole cell extract. Samples were loaded onto a 5% SDS-PAGE gel and probed with a DNMT1 polyclonal antibody (Product # PA5-30581) at a dilution of 1:500.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis was performed on modified whole cell extracts (1% SDS) (30 µg lysate) of PANC-1 (Lane 1), HeLa (Lane 2), HEK-293 (Lane 3), HCT 116 (Lane 4), PC-12 (Lane 5), SH-SY5Y (Lane 6) and NTERA-2 (Lane 7). The blot was probed with Anti-DNMT1 Polyclonal Antibody (Product # PA5-30581, 1:1000 dilution) and detected by chemiluminescence using Goat anti Rabbit IgG (H+L) Superclonal™ Secondary Antibody, HRP conjugate (Product # A27036, 0.25 µg/ml, 1:4000 dilution). A 183 kDa band corresponding to DNMT1 was detected across the cell lines tested.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot analysis of DNMT1 was performed by separating 30 µg of various whole cell extracts by 5% SDS-PAGE. Proteins were transferred to a membrane and probed with a DNMT1 Polyclonal Antibody (Product # PA5-30581) at a dilution of 1:5000 and a HRP-conjugated anti-rabbit IgG secondary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-DNMT1 Polyclonal Antibody (Product # PA5-30581) and a 183 kDa band corresponding to DNMT1 was observed. Nuclear enriched extracts (40 µg lysate) of K-562 (Lane 1), K-562 treated with 5 µM Aza deoxycytidine for 3 days (medium and drug were replaced every 24 hours) (Lane 2), MCF7 (Lane 3), HEK-293 (Lane 4) and MCF 10A (Lane 5) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein 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 (1:5000) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036,1:20,000) using the iBright™ FL1500 Imaging System (Product # A44115). Chemiluminescent detection was performed using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Product # 34580). Down-regulation of DNMT1 expression upon Aza deoxycytidine treatment in K-562, and relative expression between MCF7 and MCF 10A were observed as expected.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
Main image
Experimental details: Western Blot analysis of DNMT1 was performed by separating 30 µg of various whole cell extracts by 5% SDS-PAGE. Proteins were transferred to a membrane and probed with a DNMT1 Polyclonal Antibody (Product # PA5-30581) at a dilution of 1:5000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of DNMT1 (red) in human melanocytes. Cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.2% Triton X-100 in PBS for 10 minutes at room temperature, and blocked with 5% normal goat serum for 1 hour at room temperature. Cells were probed with a DNMT1 polyclonal antibody (Product # PA5-30581) at a dilution of 1:500 for 1 hour at room temperature, washed with PBS, and incubated with an anti-rabbit Alexa-Fluor 555-conjugated secondary antibody at a dilution of 1:500 for 1 hour at room temperature. Nuclei (blue) were stained with DAPI. Images were taken on a fluorescent microscope at 20X magnification. Data courtesy of the Innovators Program.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescent analysis of DNMT1 (red) in human melanocytes. Cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.2% Triton X-100 in PBS for 10 minutes at room temperature, and blocked with 5% normal goat serum for 1 hour at room temperature. Cells were probed with a DNMT1 polyclonal antibody (Product # PA5-30581) at a dilution of 1:500 for 1 hour at room temperature, washed with PBS, and incubated with an anti-rabbit Alexa-Fluor 555-conjugated secondary antibody at a dilution of 1:500 for 1 hour at room temperature. Nuclei (blue) were stained with DAPI. Images were taken on a fluorescent microscope at 20X magnification. Data courtesy of the Innovators Program.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunocytochemistry-Immunofluorescence analysis of DNMT1 was performed in HeLa cells fixed in 4% paraformaldehyde at RT for 15 min. Green: DNMT1 Polyclonal Antibody (Product # PA5-30581) diluted at 1:2000. Red: phalloidin, a cytoskeleton marker.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of DNMT1 was performed using 70% confluent log phase K-562 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 DNMT1 Polyclonal Antibody (Product # PA5-30581) at 1:500 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), 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 predominant nuclear localization. Panel e represents merged image of K-562 cells treated with 5 µM Aza deoxycytidine for 3 days (medium and drug were replaced every 24 hours). The signal is significantly lower in panel e due to down-regulation of DNMT1 expression upon Aza deoxycytidine treatment. Panel f represents untreated 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: Enrichment of endogenous DNMT1 protein at specific gene loci using Anti-DNMT1 Antibody: Chromatin Immunoprecipitation (ChIP) was performed using Anti-DNMT1 Rabbit Polyclonal Antibody (Product # PA5-30581, 4 ug) on sheared chromatin from 2 million HeLa 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 mir-137 and POMC (positive) and SAT2 satellite repeats (negative). Data is presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative CT method.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Cross-linked ChIP was performed with HCT116 chromatin extract and 5 µg of either control rabbit IgG or DNMT1 Polyclonal Antibody (Product # PA5-30581). The precipitated DNA was detected by PCR with primer set targeting to ILIR2.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: DNMT1 Polyclonal Antibody immunoprecipitates DNMT1 protein in IP experiments. IP samples: HeLa nuclear extract. A. Control with 4 µg of preimmune Rabbit IgG. B. Immunoprecipitation of DNMT1 protein by 4 µg DNMT1 Polyclonal Antibody (Product # PA5-30581). 5 % SDS-PAGE. The immunoprecipitated DNMT1 protein was detected by DNMT1 Polyclonal Antibody (Product # PA5-30581) diluted at 1:1,000.

PA5-30581

Antibody data