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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 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.
ICC/IF image of ab37264 stained human MCF7 cells. The cells were methanol fixed (5 min), permabilised in 0.1% PBS-Tween (20 min) and incubated with the antibody (ab37264, 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 and HEK 293 cells.