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This antibody is raised using the same immunogen as our popular anti-Histone H3 rabbit polyclonal - ab1791
Our Abpromise guarantee covers the use of ab12079 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ChIP||Use 2-4 µg for 25 µg of chromatin. Every new batch of this antibody is tested at Abcam in ChIP.|
|IP||Use at an assay dependent concentration.|
|ChIP/Chip||Use at an assay dependent concentration.|
|WB||Use a concentration of 1 µg/ml. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa).Can be blocked with Human Histone H3 peptide (ab12149).|
|IHC-P||Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.|
Chromatin was prepared from U2OS 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 ab12079 (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 for active and inactive loci, Sybr green approach for heterochromatic loci). Primers and probes are located in the first kb of the transcribed region.
IHC image of Histone H3 staining in human testis formalin fixed paraffin embedded tissue section, performed on a Leica Bond system using the standard protocol B. 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 ab12079, 5µg/ml, for 15 mins at room temperature. A donkey anti-goat biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.
For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
Histone H3 antibody (ab12079) at 0.5 ug/ml, HeLa Cell Histone prep at 5 ug
Rabbit polyclonal to Goat IgG H&L (HRP) (ab6741) at 1/5000 dilution
Performed under reducing conditions.
Predicted band size : 15kD