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Synthetic peptide conjugated to KLH derived from within residues 150 - 250 of Human macroH2A.1.
(Peptide available as ab37263.)
Our Abpromise guarantee covers the use of ab37264 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ChIP||Use at an assay dependent concentration. PubMed: 19380460|
|Flow Cyt||Use at an assay dependent concentration. ab171870-Rabbit polyclonal IgG, is suitable for use as an isotype control with this antibody.|
|WB||Use a concentration of 1 µg/ml. Detects a band of approximately 40 kDa (predicted molecular weight: 40 kDa).Can be blocked with Rat macroH2A.1 peptide (ab37263).|
|IHC-P||Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.|
|ICC/IF||Use a concentration of 1 - 5 µg/ml.|
Lane 1: Wild-type HAP1 whole cell lysate (20 µg)
Lane 2: MacroH2A.1 knockout HAP1 whole cell lysate (20 µg)
Lane 3: HeLa whole cell lysate (20 µg)
Lane 4: HepG2 whole cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab37264 observed at 40 kDa. Red - loading control, ab18058, observed at 130 kDa.
ab37264 was shown to specifically recognize macroH2A.1 in wild-type HAP1 cells along with additional cross reactive bands. No band was observed when macroH2A.1 knockout cells were examined. Wild-type and macroH2A.1 knockout samples were subjected to SDS-PAGE. Ab37264 and ab18058 (Mouse anti Vinculin loading control) were incubated overnight at 4°C at 1 ug/ml and 1/10,000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.
ab37264 stained in Hela cells. Cells were fixed with 100% methanol (5 min) 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 ab37264 at 5 µg/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 µg/ml for 1 hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43µM for 1hour at room temperature.