Anti-Histone H2B (mono methyl K5) antibody - ChIP Grade (ab12929)

  ChIP - Histone H2B (mono methyl K5) antibody - ChIP Grade (ab12929)

Chromatin was prepared from Hela 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 ab12929 (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.

  Western blot - Histone H2B (mono methyl K5) antibody (ab12929)

All lanes : Anti-Histone H2B (mono methyl K5) antibody - ChIP Grade (ab12929) at 1 µg/ml

Lane 1 : Calf Thymus Histone Preparation Nuclear Lysate (ab121)
Lane 2 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Histone H2B peptide - mono methyl K5 (ab13211)
Lane 3 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Histone H2B peptide (ab13212)

Lysates/proteins at 20 µg per lane.

Secondary
IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size : 14 kDa

  ChIP - Histone H2B (mono methyl K5) antibody - ChIP Grade (ab12929)

ab12929 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 untreated.

This image is courtesy of an anonymous abreview.

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  Immunoprecipitation - Anti-Histone H2B (mono methyl K5) antibody - ChIP Grade (ab12929)

Histone H2B (mono methyl K5) was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to Histone H2B (mono methyl K5) - ChIP Grade 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 ab12929.
Secondary: Clean blot (HRP conjugate) at 1/1000 dilution.
Band: 17kDa: Histone H2B (mono methyl K5).

  Immunocytochemistry/ Immunofluorescence - Anti-Histone H2B (mono methyl K5) antibody - ChIP Grade (ab12929)

ICC/IF image of ab12929 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab12929 at 1µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.