Anti-pan methyl Lysine antibody - ChIP Grade

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-pan methyl Lysine antibody - ChIP Grade (AB7315)

ICC/IF image of ab7315 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 ab7315 at 5μg/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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-pan methyl Lysine antibody - ChIP Grade (AB7315)

IHC image of pan methyl Lysine (methyl K pan) 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 ab7315, 1µg/ml, for 15 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.

• ChIP

Unknown

ChIP - Anti-pan methyl Lysine antibody - ChIP Grade (AB7315)

Chromatin was prepared from Hela cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min. The ChIP was performed with 25μg of chromatin, 6.5μl of ab7315 (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.

• IP

Lab

Immunoprecipitation - Anti-pan methyl Lysine antibody - ChIP Grade (AB7315)

pan methyl Lysine was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of it polyclonal to pan methyl Lysine 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 ab7315.
Secondary : Clean blot (HRP conjugate) at 1/1000 dilution.
Band : 17kDa : pan methyl Lysine.

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Immunoprecipitation - Anti-pan methyl Lysine antibody - ChIP Grade (ab7315)

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• WB

Project1592****

Western blot - Anti-pan methyl Lysine antibody - ChIP Grade (AB7315)

Expected molecular weights : H3 = 17 kDa; H4 = 14 kDa This image shows that the main epitopes recognized by ab7315 are the di methylated lysine residues. This can be seen in lanes 4, 7 and 10 where the activity of the antibody is quenched by the immunizing peptides (ab7768, ab1772, ab1781).

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Western blot - Anti-pan methyl Lysine antibody - ChIP Grade (ab7315) at 1 µg/mL

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Calf Thymus Histone Preparation Nuclear Lysate at 0.5 µg

Secondary

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Western blot - Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-preadsorbed-ab97080'>ab97080</a>) at 1/5000 dilution

Predicted band size: 14-17 kDa

true

Exposure time: 20min