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Synthetic peptide conjugated to KLH derived from within residues 1 - 100 of Human Histone H2A, symmetric di methylated at R3.
(Peptide available as ab22399.)
Our Abpromise guarantee covers the use of ab22397 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|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.|
|ICC/IF||Use a concentration of 5 µg/ml.|
|ELISA||Use at an assay dependent concentration.
This antibody gave a positive result in ELISA against the immunizing peptide (ab22399). It gave a negative result in ELISA against the non-modified equivalent peptide (ab13186). This indicates that it is specific for the modified peptide. See figure below. Not yet tested in other applications. Optimal dilutions/concentrations should be determined by the end user.
IHC image of ab22397 staining Histone H2A (symmetric di methyl R3) in human kidney formalin fixed paraffin embedded tissue sections, performed on a Leica Bond. 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 ab22397, 5µg/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. No primary antibody was used in the negative control (shown on the inset).
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.
ICC/IF image of ab22397 stained human MCF7 cells. The cells were methanol fixed (5 min), permabilised in 0.1% PBS-Tween (20 min) and incubated with the antibody (ab22397, 5µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue). This antibody also gave a positive IF result in HeLa, HEK 293 and HepG2 cells. However, this Fast-Track antibody is not yet fully characterised. This image represents inconclusive preliminary data.