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Synthetic peptide corresponding to Human Histone H3 aa 1-100 (tri methyl K4) conjugated to Keyhole Limpet Haemocyanin (KLH) (Cysteine residue).
(Peptide available as
Our Abpromise guarantee covers the use of ab1012 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|PepArr||Use a concentration of 0.5 - 2 µg/ml.|
|ChIP||Use 2-5 µg for 25 µg of chromatin.|
|Flow Cyt||Use 1µg for 106 cells.
ab170192 - Mouse monoclonal IgG2b, is suitable for use as an isotype control with this antibody.
|ICC/IF||Use at an assay dependent concentration.|
|IHC-Fr||Use at an assay dependent concentration. PubMed: 20047469|
|WB||Use a concentration of 1 - 5 µg/ml. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa).Can be blocked with Human Histone H3 (tri methyl K4) peptide (ab1342). NOT SUITABLE for blocking with milk. Block in 5% BSA for 1 hour. Our labs have investigated the blocking conditions for this antibody and found that milk significantly decreases the signal and is therefore not a suitable blocking agent for this antibody (see image below).|
|ICC||Use a concentration of 5 µg/ml.|
ELISA using ab1012 at varying antibody concentrations.
Curve_SPL1 indicates binding to the tri methyl K4 peptide ab1342. Curve_SPL4 indicates partial binding to the di methyl K4 peptide ab7768. There is very weak cross-reactivity with the mono methyl K4 peptide ab1340 (Curve_SPL3).
Binding to the following peptides was not seen: SPL2 unmodified Histone H3, SPL5 Histone H3 mono methyl K9, SPL6 Histone H3 di methyl K9, SPL7 Histone H3 tri methyl K9, SPL8 Histone H3 mono methyl K27, SPL9 Histone H3 di methyl K27, SPL10 Histone H3 tri methyl K27.
All batches of ab1012 are tested in Peptide Array against peptides to different Histone 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 - tri methyl K4 peptide (ab92374), indicating that this antibody specifically recognises the Histone H3 - tri methyl K4 modification.
|ab92374 - Histone H3 - tri methyl K4|
|ab7768 - Histone H3 - di methyl K4|
|ab179150 - Histone H3 - asymmetric di methyl R2|
|ab179142 - Histone H3 - mono methyl R2|
|ab1784 - Histone H3 - di methyl K36|
|ab179146 - Histone H3 - symmetric di methyl R2|
|ab36927 - Histone H2A - phospho S122|
|ab46854 - Histone H3 - tri methyl K18|
|ab179021 - Histone H2A - symmetric di methyl R29|
|ab17632 - Histone H4 - biotinylated K5|
|ab179159 - Histone H3 - phospho T3|
|ab14103 - Histone H3 - phopsho T6|
|ab1783 - Histone H3 - mono methyl K36|
|ab179177 - Histone H3 - mono methyl K4 + Histone H3 di methyl K9|
Chromatin was prepared from U2OS cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min. The ChIP was performed with 25 µg of chromatin, 5 µg of ab1012 (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.
ICC/IF image of ab1012 stained HeLa cells. The cells were 4% PFA fixed (10 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 (ab1012, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse 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. This antibody also gave a positive result in 4% PFA fixed (10 min) HepG2, Hek293 and MCF7 cells at 5µg/ml, and in 100% methanol fixed (5 min) HeLa, Hek293, HepG2 and MCF7 cells at 5µg/ml.
Overlay histogram showing HeLa cells stained with ab1012 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab1012, 1µg/1x106) for 30 min at 22°C. The secondary antibody used was Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) preadsorbed (ab150117) at 1/2000 dilution for 30 min at 22°C.
Isotype control antibody (black line) was Mouse IgG2b [7E10G10] isotype control (ab170192) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.
Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter.
This antibody gave a positive signal in HeLa cells fixed with 4% formaldehyde (10 min)/permeabilized with 0.1% PBS-Triton X-100 for 15 min used under the same conditions.
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