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Fusion protein, corresponding to amino acids 82-398 of Human HEF1.
Our Abpromise guarantee covers the use of ab18056 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||1/2000 - 1/6000. Predicted molecular weight: 93 kDa.|
|ICC/IF||Use at an assay dependent concentration. PubMed: 19376971|
|IHC-Fr||Use at an assay dependent concentration. PubMed: 19464348|
|IHC-P||Use at an assay dependent concentration.|
|Flow Cyt||Use 1µg for 106 cells. ab170190-Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.|
Image courtesy of an Abreview submitted by Melina Reisenberg.
ICC/IF image of ab18056 stained MCF7 (human breast adenocarcinoma cell line) cells. The cells were fixed in 4% formaldehyde for 10 minutes and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1 hour to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab18056, 1 µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse IgG (H+L) used at a 1/1000 dilution for 1 hour. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1 hour. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43 µM.
Overlay histogram showing A549 (human lung carcinoma cell line) cells stained with ab18056 (red line). The cells were fixed with 80% methanol (5 minutes) and then permeabilized with 0.1% PBS-Tween for 20 minutes. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab18056, 1 µg/ 1 x 106 cells) for 30 minutes at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 minutes at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2 µg/ 1 x 106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in A549 cells fixed with 4% paraformaldehyde (10 minutes)/permeabilized in 0.1% PBS-Tween used under the same conditions.
IHC image of ab18056 staining in human kidney formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6, epitope retrieval solution 1) for 20 minutes. The section was then incubated with ab18056, 10 µg/ml, for 15 minutes 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.
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.
ab18056 Immunoprecipitating Human Lung Adenocarcinoma A549 cell-line. 1000µg of cell lysate was incubated with primary antibody (1/1000) and matrix (Protein G) for 20 hours at 4°C. Treatment was 100ng/ml of HGF or EGF for 5 or 60 minutes.
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