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  ab2886 (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. Primers and probes are located in the first kb of the transcribed region.

Anti-Histone H3 (mono methyl K79) antibody - ChIP Grade (ab2886) at 1 µg/ml + Calf Thymus Histone Preparation Nuclear Lysate at 0.5 µg

Secondary
Human NFIB / NF1B2 peptide (ab95051) at 1/10000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 15.4 kDa
Additional bands at: 17 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 3 minutes

All batches of ab2886 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 - mono methyl K79 peptide (ab4555), indicating that this antibody specifically recognises the Histone H3 - mono methyl K79 modification.

ab4558 - Histone H3 - unmodified

ab1771 - Histone H3 - di methyl K9

ab4555 - Histone H3 - mono methyl K79

ab4556 - Histone H3 - di methyl K79

ab4557 - Histone H3 - tri methyl K79

ab4560 - Histone H4 - di methyl K79

All lanes : Anti-Histone H3 (mono methyl K79) antibody - ChIP Grade (ab2886) at 1/300 dilution

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
Lane 3 : Testis (Rat) Tissue Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (HRP), pre-adsorbed at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 15.4 kDa
Observed band size: 18 kDa why is the actual band size different from the predicted?


Exposure time: 4 minutes

Chromatin was prepared from whole cell lysate of normal rat liver and liver cancer cells. The cross-linking (X-ChiP) technique was used, crosslinking was performed for 15 minutes in formaldehyde. 5 µg of the primary antibody was used in 1/100 dilution and it was incubated with the sample for 16 hours at 4°C in a commercially available ChIP dilution buffer. The immunoprecipitated DNA was quantified by real time PCR. ChIP results show that the Histone H3 (mono methyl K79) and GAPDH genes are expressed in higher levels in liver cancer cells than in normal liver cells.

See Abreview

Histone H3 (mono methyl K79) was immunoprecipitated using 0.5mg HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate, 5µg of Rabbit polyclonal to Histone H3 (mono methyl K79) and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab2886.
Secondary: Clean blot (HRP conjugate) at 1/1000 dilution.
Band: 18kDa: Histone H3 (mono methyl K79).