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H3K79me3 Polyclonal Antibody

Human osteosarcoma (U2OS) cells were stained with the Bioss antibody against H3K79me3 (Cat. No. bs-53121R) and with DAPI. Cells were fixed with 2.5% formaldehyde for 30\u2019 and blocked with PBS\/TX-100 containing 1% BSA. Figure A: cells were immunofluorescently labeled with the H3K79me3 antibody (left) diluted 1:300 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa568 or with DAPI (right). Figure B and C: staining of the cells with the H3K79me3 antibody after incubation of the antibody with 2 ng\/\u03bcl H4K20me3 and H3K79me3 peptide, respectively.
Human osteosarcoma (U2OS) cells were stained with the Bioss antibody against H3K79me3 (Cat. No. bs-53121R) and with DAPI. Cells were fixed with 2.5% formaldehyde for 30\u2019 and blocked with PBS\/TX-100 containing 1% BSA. Figure A: cells were immunofluorescently labeled with the H3K79me3 antibody (left) diluted 1:300 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa568 or with DAPI (right). Figure B and C: staining of the cells with the H3K79me3 antibody after incubation of the antibody with 2 ng\/\u03bcl H4K20me3 and H3K79me3 peptide, respectively.
Histone extracts of HeLa cells (15 \u03bcg) were analyzed by Western blot using the Bioss antibody against H3K79me3 (Cat. No. bs-53121R), diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The molecular weight marker is shown on the right; the location of the protein of interest is indicated on the left.
Histone extracts of HeLa cells (15 \u03bcg) were analyzed by Western blot using the Bioss antibody against H3K79me3 (Cat. No. bs-53121R), diluted 1:1,000 in TBS-Tween containing 5% skimmed milk. The molecular weight marker is shown on the right; the location of the protein of interest is indicated on the left.
A Dot Blot analysis was performed to test the cross-reactivity of the Bioss antibody against H3K79me3 (Cat. No. bs-53121R) with peptides containing other histone modifications and the unmodified H3K79. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:20,000.The figure shows a high specificity of the antibody for the modification of interest.
A Dot Blot analysis was performed to test the cross-reactivity of the Bioss antibody against H3K79me3 (Cat. No. bs-53121R) with peptides containing other histone modifications and the unmodified H3K79. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1:20,000.The figure shows a high specificity of the antibody for the modification of interest.
To determine the titer of the antibody, an ELISA was performed using a serial dilution of Bioss antibody directed against H3K79me3 (Cat. No. bs-53121R), crude serum and flow through in antigen-coated wells. The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:3,500.
To determine the titer of the antibody, an ELISA was performed using a serial dilution of Bioss antibody directed against H3K79me3 (Cat. No. bs-53121R), crude serum and flow through in antigen-coated wells. The antigen used was a peptide containing the histone modification of interest. By plotting the absorbance against the antibody dilution, the titer of the antibody was estimated to be 1:3,500.
ChIP was performed with the Bioss antibody against H3K79me3 (Cat. No. bs-53121R) on sheared chromatin from 1 million HeLaS3 cells using an Auto Histone ChIP-seq kit and the SX-8G IP-Star automated system. A titration of the antibody consisting of 1, 2, 5 and 10 \u03bcg per ChIP experiment was analyzed. IgG (5 \u03bcg\/IP) was used as negative IP control. Quantitative PCR was performed with primers for the GAPDH promoter and for exon 2 of the inactive myoglobin gene. The figure shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis). These results are in accordance with the observation that H3K79me3 shows a preference for active promoters.
ChIP was performed with the Bioss antibody against H3K79me3 (Cat. No. bs-53121R) on sheared chromatin from 1 million HeLaS3 cells using an Auto Histone ChIP-seq kit and the SX-8G IP-Star automated system. A titration of the antibody consisting of 1, 2, 5 and 10 \u03bcg per ChIP experiment was analyzed. IgG (5 \u03bcg\/IP) was used as negative IP control. Quantitative PCR was performed with primers for the GAPDH promoter and for exon 2 of the inactive myoglobin gene. The figure shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis). These results are in accordance with the observation that H3K79me3 shows a preference for active promoters.
ChIP was performed as described above using 2 \u03bcg of the Bioss antibody against H3K79me3 (bs-53121R). The IP\u2019d DNA was analyzed on an Illumina Genome Analyzer. Library preparation, cluster generation, and sequencing were performed according to the manufacturer\u2019s instructions. The 36 bp tags were aligned to the human genome using the ELAND algorithm. The figure shows the peak distribution along the complete sequence and a 600 kb region of the X-chromosome (figure A and B), in a 2 Mb region from chromosome 11 (figure C), and in a 100 kb region surrounding the GAPDH positive control gene (figure D).
ChIP was performed as described above using 2 \u03bcg of the Bioss antibody against H3K79me3 (bs-53121R). The IP\u2019d DNA was analyzed on an Illumina Genome Analyzer. Library preparation, cluster generation, and sequencing were performed according to the manufacturer\u2019s instructions. The 36 bp tags were aligned to the human genome using the ELAND algorithm. The figure shows the peak distribution along the complete sequence and a 600 kb region of the X-chromosome (figure A and B), in a 2 Mb region from chromosome 11 (figure C), and in a 100 kb region surrounding the GAPDH positive control gene (figure D).
Bioss unconjugated antibody vial

Applications

  • WB
  • ELISA
  • IF
  • dot-blot

Reactivity

  • Human
  • Mouse
  • Yeast
Overview
Catalog # bs-53121R-50ug
Product Name H3K79me3 Polyclonal Antibody
Applications
WB ELISA IF DOT-BLOT
Reactivity Human, Mouse, Yeast
Specifications
Conjugation Unconjugated
Host Rabbit
Source Polyclonal antibody raised in rabbit against histone H3 containing the trimethylated lysine 79 (H3K79me3), using a KLH-conjugated synthetic peptide
Clonality Polyclonal
Isotype Not Determined
Concentration 0.75ug/ul
Purification Affinity purified polyclonal antibody in PBS containing 0.05% azide and 0.05% ProClin 300.
Storage Buffer 0.01M TBS(pH7.4) with 1% BSA, 0.02% Proclin300 and 50% Glycerol.
Storage Condition Store at -20C for 12 months.
Target
Gene ID 8350
Swiss Prot P68431
Background Core component of nucleosome. Nucleosomes wrap and compact DNA into chromatin, limiting DNA accessibility to the cellular machineries which require DNA as a template. Histones thereby play a central role in transcription regulation, DNA repair, DNA replication and chromosomal stability. DNA accessibility is regulated via a complex set of post-translational modifications of histones, also called histone code, and nucleosome remodeling.
Application Dilution
WB 1:300-5000
ELISA 1:500-1000
IF
dot-blot

Frequently asked questions

Which applications is H3K79me3 Polyclonal Antibody (bs-53121R) validated for, and at what dilution?

bs-53121R is validated for WB (1:300-5000); ELISA (1:500-1000); IF; dot-blot. These are recommended starting dilutions; optimal dilutions should be determined by the end user.

Which species does H3K79me3 Polyclonal Antibody (bs-53121R) react with?

Tested reactivity: Human, Mouse, Yeast.

How should bs-53121R be stored?

Store at -20C for 12 months. Storage buffer: 0.01M TBS(pH7.4) with 1% BSA, 0.02% Proclin300 and 50% Glycerol.

What immunogen was used to raise H3K79me3 Polyclonal Antibody (bs-53121R)?

Polyclonal antibody raised in rabbit against histone H3 containing the trimethylated lysine 79 (H3K79me3), using a KLH-conjugated synthetic peptide

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