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Synthetic peptide derived from residues 1 - 100 of Human Histone H1.2.
Histone H1.2 is one of five known h1 variants (known as H1.1/2/3/4/5 and/or H1.a/b/c/d/e). The H1 variants differ from each other in the amino acid sequence of their N-terminal regions.
Our Abpromise guarantee covers the use of ab4086 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ChIP||Use 2-6 µg for 25 µg of chromatin.|
|WB||1/500. Detects a band of approximately 25 kDa (predicted molecular weight: 21 kDa).Can be blocked with Human Histone H1.2 peptide (ab16936).|
|IP||Use a concentration of 5 µg/ml.|
|ICC/IF||Use a concentration of 1 µg/ml.|
|IHC-P||Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.|
Histone H1.2 - 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 ab4086.
Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band: 21kDa; Histone H1.2 - ChIP Grade
ab4086 Histone H1.2
The antibody was used at a dilution of 1/500
Lane 1: HeLa Histone (5ug) + ab4086
Lane 2: HeLa Histone (5ug) + ab4086 + 1
Secondary ab: Goat polyclonal to Rabbit IgG H&L (HRP) Pre-Adsorbed ab7090 (1/5000)
Exposure time: 1 minute
Expected molecular weight: 21.3 kDa
ICC/IF image of ab4086 stained human HeLa cells. The cells were PFA fixed (3.7% PFA, 10 min) and incubated with the antibody (ab4086, 1µg/ml) for 1h at room temperature. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Image-iTTM FX Signal Enhancer was used as the primary blocking agent, 5% BSA (in TBS-T) was used for all other blocking steps. DAPI was used to stain the cell nuclei (blue). Alexa Fluor® 594 WGA was used to label plasma membranes (red).
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, 2µg of ab4086 (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.
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