Western blot analysis of extracts from HCT 116, K-562, and COS-7 cells using HIRA (D2A5E) Rabbit mAb.
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Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. 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
HIRA (D2A5E) Rabbit mAb recognizes endogenous levels of total HIRA protein.Species Reactivity:
Human, Mouse, Monkey
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu593 of human HIRA protein.
Histone cell cycle regulation defective homolog A (HIRA), also known as TUP1-like enhancer of split protein 1 (TUPLE1), is the mammalian homolog of the yeast HIR1 and HIR2 transcriptional repressor proteins (1). HIRA interacts with UBN1, CABIN, and ASF1A in the cell nucleus to form the evolutionarily conserved HUCA histone chaperone complex that deposits the variant histone H3.3 into chromatin in a DNA-replication independent manner (2). HIRA is required for deposition of histone H3.3 at the transcription start sites of genes, where incorporation of histone H3.3 facilitates nucleosome destabilization and contributes to transcriptional activation (3-5). Histone H3.3 is also linked to gene silencing and is incorporated into regions of the genome thought to be transcriptionally inactive (5-7). While some incorporation of H3.3 into heterochromatin is facilitated by a different histone chaperone complex that contains ATRX and DAXX (ie. telomeric incorporation of H3.3), HIRA is required for incorporation of histone H3.3 and formation of senescence-associated heterochromatin foci (SAHF) during cellular senescence (5-8). HIRA is ubiquitously expressed during mouse embryonic development (9). In the adult mouse, HIRA is expressed at high levels in the kidney, skeletal muscle, and pancreas, but it is expressed at lower levels in the heart, lung, placenta, brain, and liver (9). A missing copy of the HIRA gene on human chromosome region 22q11.2 is a common characteristic of DiGeorge syndrome patients and insufficient production of the HIRA protein may disrupt normal embryonic development (9).
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