Immunocytochemistry/ Immunofluorescence - Histone H3 (mono methyl K79) antibody - ChIP Grade (ab2886)

This image was kindly supplied as part of the review submitted by Dr Geza Fejes-Toth.

Western blot

All lanes : Anti-Histone H3 (mono methyl K79) antibody - ChIP Grade (ab2886) at 1/500 dilution

Lane 1 : Calf thymus histone lysate
Lane 2 : Calf thymus histone lysate with Histone H3 peptide - mono methyl K79 (ab4555) at 1 µg/ml
Lane 3 : Calf thymus histone lysate with Histone H3 peptide - di methyl K79 (ab4556) at 1 µg/ml
Lane 4 : Calf thymus histone lysate with Histone H3 peptide - tri methyl K79 (ab4557) at 1 µg/ml
Lane 5 : Calf thymus histone lysate with Histone H4 peptide - di methyl K79 (ab4560) at 1 µg/ml
Lane 6 : Calf thymus histone lysate with Histone H3 peptide - mono methyl K9 (ab1771) at 1 µg/ml
Lane 7 : Calf thymus histone lysate with Unmodified K79 of Histone H3 peptide (ab4264) at 1 µg/ml

Secondary
Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (HRP) (ab6721) at 1/5000 dilution

Predicted band size : 15.4 kDa
Observed band size : 17 kDa (why is the actual band size different from the predicted?)
Additional bands at : 14 kDa. We are unsure as to the identity of these extra bands.

Exposure time : 5 minutes

ab2886 specifically detects Histone H3 mono methyl K79. As can be seen in Lane 2, the addition of the blocking peptide (ab4555) quenches the activity of the antibody.

ChIP - Histone H3 (mono methyl K79) antibody - ChIP Grade (ab2886)

Chromatin was prepared from U2OS 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.

Western blot - Histone H3 (mono methyl K79) antibody - ChIP Grade (ab2886)

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
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

ChIP - Histone H3 (mono methyl K79) antibody - ChIP Grade (ab2886)

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.

This image is a courtesy of Anonymous Abreview

Immunoprecipitation - Anti-Histone H3 (mono methyl K79) antibody - ChIP Grade (ab2886)

Histone H3 (mono methyl K79) was immunoprecipitated using 0.5mg Hela whole cell extract, 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).