All lanes : Anti-Histone H2B (acetyl K5) antibody [EP857Y] - ChIP Grade (ab40886) at 1/10000 dilution (Purified)

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates
Lane 2 : HeLa (Human cervix adenocarcinoma epithelial cell) treated with 500ng/ml trichostatin A for 4 hours whole cell lysates

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 14 kDa
Observed band size: 14 kDa

Blocking/Diluting Buffer and concentration: 5% NFDM/TBST

Chromatin was prepared from HeLa  (Human cervix adenocarcinoma epithelial cell) cells according to the Abcam X-ChIP protocol*. Cells were fixed with formaldehyde for 10 minutes.
The ChIP was performed with 25 µg of chromatin, 2µg of ab40886 (red), or 2 µg of rabbit normal IgG (gray) and 20 µl of Protein A/G sepharose beads. The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci, Sybr green approach for heterochromatic loci).
Primers and probes are located in the first kb of the transcribed region.
*http://www.abcam.com/resources?keywords=X%20ChIP%20protocol

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human hepatocellular carcinoma tissue sections labeling Histone H2B with purified ab40886 at 1/100 dilution (5.42 µg/mL). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody.

Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) treated with 500 ng/mL TSA for 4 hours cells labeling Histone H2B with purified ab40886 at 1/1000 dilution (0.54 µg/mL). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 µg/mL). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 µg/mL) dilution. DAPI (blue) was used as nuclear counterstain.

PBS instead of the primary antibody was used as the secondary antibody only control.

All lanes : Anti-Histone H2B (acetyl K5) antibody [EP857Y] - ChIP Grade (ab40886) at 1/10000 dilution (Purified)

Lane 1 : NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates
Lane 2 : NIH/3T3 (Mouse embryonic fibroblast) treated with 500ng/ml trichostatin A for 4 hours whole cell lysates

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 14 kDa
Observed band size: 14 kDa

Blocking/Diluting Buffer and concentration: 5% NFDM/TBST

All lanes : Anti-Histone H2B (acetyl K5) antibody [EP857Y] - ChIP Grade (ab40886) at 1/10000 dilution (Purified)

Lane 1 : C6 (Rat glial tumor glial cell) whole cell lysates
Lane 2 : C6 (Rat glial tumor glial cell) treated with 500ng/ml trichostatin A for 4 hours whole cell lysates

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 14 kDa
Observed band size: 14 kDa

Blocking/Diluting Buffer and concentration: 5% NFDM/TBST

Chromatin was prepared from HeLa (Human cervix adenocarcinoma epithelial cell) cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min. The  ChIP was performed with 25µg of chromatin, 6µl of unpurified ab40886 (blue), and 20µl 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 for active and inactive loci, Sybr green approach for heterochromatic loci). Primers and probes are located in the first kb of the transcribed region.    

Anti-Histone H2B (acetyl K5) antibody [EP857Y] - ChIP Grade (ab40886) at 1/3000 dilution + Drosophila melanogaster lysate (Fruit fly larvae) at 20 µg

Secondary
Anti-rabbit IgG HRP at 1/6000 dilution

Predicted band size: 14 kDa

See Abreview

ab40886 (unpurified) staining human bladder carcinoma for Histone H2B expression (1/250 dilution)

Heat mediated antigen retrieval was performed with citrate buffer pH 6 before commencing with IHC staining protocol.

ab40886 (unpurified) (1/250) staining A431 (Human epidermoid carcinoma cell line) cells by immunofluorescence.

ab40886 (unpurified) at a 1/600 dilution for ChIP analysis of mouse dorsal skin epidermis whole tissue lysate, incubated for 15 hours at 4°C with ChIP dilution buffer. Cross-linking (X-ChIP) using 1% formaldehyde for 10 minutes.Detection step: Semiquantitative PCR.Negative control: Rabbit IgG.Cells treated with active vitamin D3.

See Abreview