Anti-Histone H2A antibody - ChIP Grade

• Supplier: Abcam
• Catalog: ab18255
• Prices: $404.00
• Sizes: 100 µg
• Host: Rabbit
• Clonality: Polyclonal
• Isotype: IgG
• Applications: ICC/IF ICC/IF IHC-P WB IP ChIP
• Species Reactivities: Mouse, Human
• Antigen: Synthetic peptide conjugated to KLH derived from within residues 100 to the C-terminus of Human Histone H2A
• Blocking Peptide: Human Histone H2A peptide
• Description: Rabbit Polyclonal
• Gene: HIST1H2AE
• Conjugate: Unconjugated

Product images

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 25µg of chromatin, 6µl of ab18255 (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.

Histone H2A - ChIP Grade was immunoprecipitated using 0.5mg HeLa whole cell extract, 5µg of Rabbit polyclonal to 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°C; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab18255.Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).Band: 14kDa, non specific bands - 42kDa: We are unsure as to the identity of this extra band; Histone H2A - ChIP Grade

All lanes : Anti-Histone H2A antibody - ChIP Grade (ab18255) at 1 µg/mlLane 1 : HeLa lysate at 20 µgLane 2 : HeLa nuclear lysate at 20 µgLane 3 : Calf thymus histone lysate at 20 µgLane 4 : HeLa lysate at 1 µg/ml with Human Histone H2A peptide (ab19751) at 1 µg/mlLane 5 : HeLa nuclear lysate at 1 µg/ml with Human Histone H2A peptide (ab19751) at 1 µg/mlLane 6 : Calf thymus histone lysate at 1 µg/ml with Human Histone H2A peptide (ab19751) at 1 µg/ml

Image courtesy of Human Protein Atlas ab18255 staining histone H2A in human testis, showing a distinct and strong nuclear staining pattern at cells in ductus seminiferus. Paraffin embedded human skin tissue was incubated with ab18255 (1/1200 dilution) for 30 mins at room temperature. Antigen retrieval was performed by heat induction in citrate buffer pH 6. ab18255 was tested in a tissue microarray (TMA) containing a wide range of normal and cancer tissues as well as a cell microarray consisting of a range of commonly used, well characterised human cell lines. Further results for this antibody can be found at www.proteinatlas.org.

All lanes : Anti-Histone H2A antibody - ChIP Grade (ab18255) at 1 µg/mlLane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µgLane 2 : Calf Thymus Histone Preparation Nuclear Lysate at 0.5 µgLane 3 : Histone H2A Recombinant Protein at 0.1 µgLane 4 : Histone H3.1 Recombinant Protein at 0.1 µgLane 5 : Histone H4 Recombinant Protein at 0.1 µgSecondaryGoat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilutiondeveloped using the ECL techniquePerformed under reducing conditions.

ICC/IF image of ab18255 stained HeLa cells. The cells were 100% methanol 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 (ab18255, 1µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabiit 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) HeLa, Hek293, HepG2 and MCF7 cells at 1µg/ml, and in 100% methanol fixed (5 min) Hek293, HepG2 and MCF7 cells at 1µg/ml.

All lanes : Anti-Histone H2A antibody - ChIP Grade (ab18255) at 1/1000 dilutionLane 1 : Native recombinant octamers K562 cellsLane 2 : Recombinant Human octamers containing H2ALane 3 : Recombinant Human octamers containing H2A.Z.2.1Lane 4 : Recombinant Human octamers containing H2A.Z.1Lysates/proteins at 0.5 µg per lane.SecondaryHRP-conjugated donkey anti-rabbit IgG polyclonal at 1/10000 dilutiondeveloped using the ECL techniquePerformed under reducing conditions.

Product References

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