Western blot analysis of extracts from various cell lines using MacroH2A1 Antibody.
Western blot analysis of lysates from 293 cells, mock transfected (-) or transfected with a construct expressing Myc/DDK-tagged full-length human MacroH2A1.1 (hMacroH2A1.1-Myc/DDK; +), Myc/DDK-tagged full-length human MacroH2A1.2 (hMacroH2A1.2-Myc/DDK; +), or Myc/DDK-tagged full-length human MacroH2A2 (hMacroH2A2-Myc/DDK; +), using MacroH2A1 Antibody (upper) or DYKDDDDK Tag (9A3) Mouse mAb #8146 (lower).
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Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
NOTE: Please refer to primary antibody datasheet or product webpage for recommended antibody dilution.
From sample preparation to detection, the reagents you need for your Western Blot are now in one convenient kit: #12957 Western Blotting Application Solutions Kit
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.
Load 20 µl onto SDS-PAGE gel (10 cm x 10 cm).
NOTE: Volumes are for 10 cm x 10 cm (100 cm2) of membrane; for different sized membranes, adjust volumes accordingly.
* Avoid repeated exposure to skin.
posted June 2005
revised November 2013
Reprobing of an existing membrane is a convenient means to immunoblot for multiple proteins independently when only a limited amount of sample is available. It should be noted that for the best possible results a fresh blot is always recommended. Reprobing can be a valuable method but with each reprobing of a blot there is potential for increased background signal. Additionally, it is recommended that you verify the removal of the first antibody complex prior to reprobing so that signal attributed to binding of the new antibody is not leftover signal from the first immunoblotting experiment. This can be done by re-exposing the blot to ECL reagents and making sure there is no signal prior to adding the next primary antibody.
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
posted June 2005
revised October 2016
Protocol Id: 10
MacroH2A1 Antibody recognizes endogenous levels of total MacroH2A1 protein, both isoform 1 (macroH2A1.1) and isoform 2 (macroH2A1.2). This antibody does not cross-react with MacroH2A2 protein.Species Reactivity:
Human, Mouse, Rat, MonkeySpecies predicted to react based on 100% sequence homology:
Hamster, Bovine, Guinea Pig
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Val257 of human MacroH2A1 protein. Antibodies are purified by protein A and peptide affinity chromatography.
Histone macroH2A1 and macroH2A2 comprise a family of variant histone H2A proteins. MacroH2A1 exists as two distinct isoforms due to alternative splicing of a single gene; macroH2A1.1 levels accumulate throughout differentiation and development while macroH2A1.2 shows a constant level of expression (1). MacroH2A1 and macroH2A2 are encoded by completely distinct genes located on separate chromosomes (2,3). Both macroH2A1 and macroH2A2 proteins contain an amino-terminal histone-like region with 64% sequence identity to canonical histone H2A, in addition to a carboxy-terminal “macro” domain (1-3). MacroH2A1 and macroH2A2 are enriched in facultative heterochromatin, including inactivated X chromosomes in mammalian females and senescence-associated heterochromatin foci (2-5). Both act to repress gene transcription by inhibiting the binding of transcription factors to chromatin, the acetylation of histones by p300, and the chromatin-remodeling activities of SWI/SNF and ACF (6,7). The macro domain of macroH2A1.1 binds to ADP-ribose and functions to recruit macroH2A1.1 to activated PARP at sites of DNA damage, where it mediates chromatin rearrangements to locally regulate the DNA damage response (8). MacroH2A1.2 and macroH2A2 do not bind poly-ADP-ribose and are not recruited to sites of activated PARP (8).
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