|H M R Mk||Endogenous||37||Rabbit IgG|
For western blots, incubate membrane with diluted primary antibody in 5% w/v nonfat dry milk, 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.
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 June 2016
Protocol Id: 263
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.
WDR5 (D3X5B) Rabbit mAb recognizes endogenous levels of total WDR5 protein.
Human, Mouse, Rat, Monkey
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Pro30 of human WDR5 protein.
The Set1 histone methyltransferase protein was first identified in yeast as part of the Set1/COMPASS histone methyltransferase complex, which methylates histone H3 at Lys4 and functions as a transcriptional co-activator (1). While yeast contain only one known Set1 protein, six Set1-related proteins exist in mammals: SET1A, SET1B, MLL1, MLL2, MLL3, and MLL4, all of which assemble into COMPASS-like complexes and methylate histone H3 at Lys4 (2,3). These Set1-related proteins are each found in distinct protein complexes, all of which share the common subunits WDR5, RBBP5, ASH2L, CXXC1 and DPY30. These subunits are required for proper complex assembly and modulation of histone methyltransferase activity (2-6). MLL1 and MLL2 complexes contain the additional protein subunit, menin (6). Like yeast Set1, all six Set1-related mammalian proteins methylate histone H3 at Lys4 (2-6). MLL translocations are found in a large number of hematological malignancies, suggesting that Set1/COMPASS histone methyltransferase complexes play a critical role in leukemogenesis (6).
WDR5 is a core subunit of all SET1/MLL histone methyltransferase complexes and is required for proper complex assembly and histone methyltransferase activity (7). It functions as an effector of histone H3 Lys4 methylation by recruiting SET1/MLL complexes to target loci and presenting the histone H3 amino-terminal tail for methylation (8). WDR5 contains a classical, seven-bladed WD40 propeller domain with a central cavity that binds to histone H3 Arg2 when symmetrically di-methylated (H3Arg2Me2-S) by arginine methyltransferases PRMT5 and PRMT7 (8). WDR5 binding to H3Arg2Me2-S results in increased recruitment of SET1/MLL complexes and methylation of histone H3 Lys4 at gene promoters and distal regulatory sites. In contrast, asymmetric di-methylation of histone H3 Arg2 (H3Arg2Me2-A) by PRMT6 reduces WDR5 binding and results in decreased recruitment of SET1/MLL complexes and reduced histone H3 Lys4 methylation (8). Interestingly, the H3Arg2Me2-S binding pocket of WDR5 also interacts with the SET domains of SET1/MLL proteins with comparable affinity, setting up a potential competition for WDR5 binding that may act to regulate SET1/MLL recruitment and subsequent H3 Lys4 methylation (9-11). WDR5 is also a core subunit of the ATAC and MOF-NSL histone acetyltransferase complexes and the CHD8 chromatin-remodeling complex (12-14).
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|13073S||100 µl (10 western blots)||$255.00.0|