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Synthetic peptide conjugated to KLH derived from within residues 150 to the C-terminus of Human HMGB1.
(Peptide available as ab18650.)
Our Abpromise guarantee covers the use of ab18256 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|IHC-FoFr||Use at an assay dependent concentration.|
|ICC/IF||Use a concentration of 1 µg/ml.|
|IHC-P||1/1000. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.|
|ChIP||Use at an assay dependent concentration.|
|ICC||Use at an assay dependent concentration.|
|ELISA||Use at an assay dependent concentration.|
|IP||Use at an assay dependent concentration.|
|WB||Use a concentration of 1 µg/ml. Detects a band of approximately 29 kDa (predicted molecular weight: 25 kDa).|
ab18256 stained in Hela cells. Cells were fixed with 4% paraformaldehyde (10min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% triton for 1h at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab18256 at 1µg/ml and ab7291 (Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control) at 1/1000 dilution overnight at +4°C. The secondary antibodies were ab150120 (pseudo-colored red) and ab150081 (colored green) used at 1 ug/ml for 1hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43µM for 1hour at room temperature
Paraffin embedded sections of human liver were incubated with ab18256 (1/1000 dilution) for 30 minutes at room temperature. Heat induced antigen retrieval was performed in citrate buffer pH 6.
ab18256 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 images can be found www.proteinatlas.org
ab18256 staining HMGB1 in mouse liver tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with formaldehyde and blocked with 15% serum for 60 minutes at 20°C; antigen retrieval was by heat mediation. Samples were incubated with primary antibody (1/1000 in TBS) for 18 hours at 20°C. An Alexa Fluor® 647-conjugated goat anti-rabbit IgG polyclonal (1/500) was used as the secondary antibody.
This image is courtesy of an Abreview submitted by Dr Francesca Cerbai