Western blot analysis of extracts from various cell lines using ASXL1 (D1B6V) 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
ASXL1 (D1B6V) Rabbit mAb recognizes endogenous levels of total ASXL1 protein.Species Reactivity:
Human, Mouse, Rat, Monkey
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro1330 of human ASXL1 protein.
Additional sex combs-like protein 1 (ASXL1) is a polycomb-associated protein that interacts with polycomb repressive complex 2 (PRC2), which contains the histone methyltransferase EZH2 and functions to mono-, di-, and tri-methylate histone H3 on lysine 27. These histone marks are associated with transcriptional repression (1). In addition, ASXL1 interacts with BRCA1-associated protein 1 (BAP1), the catalytic subunit of the polycomb repressive deubiquitinase complex (PR-DUB), which functions to de-ubiquitinate histone H2A at lysine 119 and activate transcription (2). ASXL1 functions as a transcriptional regulator of adipogenesis, acting to repress peroxisome proliferator-activated receptor gamma (PPARG) adipocyte differentiation. ASXL1 also functions as a transcriptional regulator of hematopoiesis acting as an activator of retinoic acid receptor (RAR) mediated transcriptional activation (3,4). ASXL1 is mutated or deleted in 10 to 30 percent of all myeloid malignancies, with loss-of-function mutations associated with poor prognosis in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Hematopoietic-specific deletions of ASXL1 in mice result in progressive, multi-lineage cytopenias and dysplasias, leading to increased number of hematopoietic stem and progenitor cells (5).
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