Western blot analysis of extracts from various cell lines using PHF6 Antibody.
|REACTIVITY||H M R Mk|
|MW (kDa)||45 (human/monkey), 41 (mouse/rat)|
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
PHF6 Antibody recognizes endogenous levels of total PHF6 protein.Species Reactivity:
Human, Mouse, Rat, Monkey
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Gly63 of human PHF6 protein. Antibodies are purified by protein A and peptide affinity chromatography.
Plant homeodomain-like finger 6 (PHF6) is a 41 kDa transcriptional repressor that was first identified as a mutated gene in Börjeson-Forssman-Lehmann syndrome (BFLS), an X-linked intellectual disability disorder (1,2). Somatic loss-of-function mutations in the PHF6 gene have also been linked to T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML) (3-5). Structurally, PHF6 contains two nuclear localization sequences, one nucleolar localization sequence, and two plant homeodomain (PHD)-like zinc fingers (6,7). Unlike other PHD proteins, the PHD domains of PHF6 are considered to be imperfect and have not been shown to directly bind to histones; however, the isolated second PHD domain (PHD2) has been shown to bind dsDNA directly (7). A more recent study finds that PHF6 interacts with histones via protein-protein interactions, and that this association is independent of DNA and enriched in the presence of the activating marks H3K27ac and H3K4me3 (8). PHF6 interacts with PAF1 and other subunits of the PAF1 transcription elongation complex, and knockdown of either PHF6 or PAF1 adversely affects proper neuronal positioning and migration in mouse cerebral cortex (9). PHF6 has also been shown to associate with members of the nucleosome remodeling and deacetylation (NuRD) chromatin remodeling complex, including CHD4, HDAC1, and RBBP4, where it is likely involved in transcriptional repression of developmental genes (10). PHF6 plays a critical role in regulating hematopoiesis, particularly by regulating chromatin accessibility to lineage-specific transcription factors. Studies suggest that PHF6 promotes B-cell lineage differentiation through the expression of B-cell specific genes, while simultaneously suppressing T-cell lineage differentiation (8). Indeed, a CRISPR-Cas9 knockout study shows that PHF6 is required for growth of B-ALL cells, while mice transplanted with PHF6-deficient B-ALL cells develop T-ALL phenotypes (8).
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