Anti-Histone H3 (di methyl K79) antibody - ChIP Grade

• WB

Lab

Western blot - Anti-Histone H3 (di methyl K79) antibody - ChIP Grade (AB3594)

Gel type : MES

Blocking buffer : 2% BSA

All lanes:

Western blot - Anti-Histone H3 (di methyl K79) antibody - ChIP Grade (ab3594) at 1 µg/mL

Lane 1:

CTH (Calf Thymus Histone) at 0.5 µg

Lane 2:

NIH 3T3 nuclear lysate (Triton enriched) at 10 µg

Secondary

All lanes:

Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/50000 dilution

Predicted band size: 15 kDa

Observed band size: 17 kDa

false

Exposure time: 1min

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (di methyl K79) antibody - ChIP Grade (AB3594)

IHC image of Histone H3 (di methyl K79) staining in human breast carcinoma FFPE section, performed on a Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab3594, 1μg/ml, for 8 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (di methyl K79) antibody - ChIP Grade (AB3594)

ab3594 staining Histone H3 (Di Methyl K79) in HeLa cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab3594 at 0.1µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• ChIP

AbReview36820****

ChIP - Anti-Histone H3 (di methyl K79) antibody - ChIP Grade (AB3594)

ChIP analysis using ab3594 binding Histone H3 in HeLa (Human epithelial adenocarcinoma cell line) cell nuclear lysate. Cells were cross-linked for 10 minutes with formaldehyde. Samples were incubated with primary antibody (5μg/μg chromatin) for 12 hours at 4°C. Protein binding was detected using real-time PCR.
Positive control : 5'UTR of transcribed gene.
Negative Control : Intergenic region.

This image is courtesy of an anonymous Abreview

• ChIP

Unknown

ChIP - Anti-Histone H3 (di methyl K79) antibody - ChIP Grade (AB3594)

Chromatin was prepared from U-2 OS (Human bone osteosarcoma epithelial cell line) cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 min. The ChIP was performed with 25 μg of chromatin, 2 μg of ab3594 (blue), and 20 μl of protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach). Primers and probes are located in the first kb of the transcribed region.

• WB

AbReview38950****

Western blot - Anti-Histone H3 (di methyl K79) antibody - ChIP Grade (AB3594)

All lanes:

Western blot - Anti-Histone H3 (di methyl K79) antibody - ChIP Grade (ab3594) at 20 µg

All lanes:

HeLa cell lysate at 20 µg

Secondary

All lanes:

HRP-conjugated Goat anti-rabbit IgG polyclonal at 1/2000 dilution

Predicted band size: 15 kDa

Observed band size: 15 kDa

true

Exposure time: 1min

This image is courtesy of an anonymous Abreview

• ICC/IF

PubMed

Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (di methyl K79) antibody - ChIP Grade (AB3594)

Verification of H3K79me2 antibody specificity.

The commercial H3K79me2 primary antibody was preincubated with (+) or without (−) antigen peptide (Abcam, catalog no. ab4556, v/v = 5 : 1) at room temperature for 1.5 h before the incubation with IVF porcine blastocysts. H3K79me2 signals were observed in blastocysts using unabsorbed primary antibody. By contrast, H3K79me2 signals were absent in blastocysts using pre-absorbed primary antibody. H3K79me2 antibody was localized with Alexa Flour 488-conjugated secondary antibody (green). DNA was stained with propidium iodide (red). Bottom panels showed the merged images (yellow) between H3K79me2 signals (green) and DNA staining (red). Scale bars = 50μm.

Tao J. et al PLoS One. 2017 Jun 20;12(6):e0179436. doi: 10.1371/journal.pone.0179436. eCollection 2017. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

• WB

Lab

Western blot - Anti-Histone H3 (di methyl K79) antibody - ChIP Grade (AB3594)

All lanes:

Western blot - Anti-Histone H3 (di methyl K79) antibody - ChIP Grade (ab3594) at 1 µg/mL

All lanes:

Calf Thymus Histone Preparation Nuclear Lysate (<a href='/en-us/products/unavailable/calf-thymus-histone-preparation-nuclear-lysate-ab121'>ab121</a>) at 0.5 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/10000 dilution

Predicted band size: 15 kDa

Observed band size: 17 kDa

true

Exposure time: 30s

• PepArr

Lab

Peptide Array - Anti-Histone H3 (di methyl K79) antibody - ChIP Grade (AB3594)

All batches of ab3594 are tested in Peptide Array against peptides to different Histone H3 modifications. Six dilutions of each peptide are printed on to the Peptide Array in triplicate and results are averaged before being plotted on to a graph. Results show strong binding to Histone H3 - di methyl K79 peptide (ab4556), indicating that this antibody specifically recognises the Histone H3 - di methyl K79 modification.

ab4556 - Histone H3 - di methyl K79

ab4555 - Histone H3 - mono methyl K79

ab4557 - Histone H3 - tri methyl K79

ab4560 - Histone H4 - di methyl K79

ab1772 - Histone H3 - di methyl K9

ab4558 - Histone H3 - unmodified