Western blot analysis of extracts from MCF7, HCT 116, and K-562 cells using MLL3 (D1S1V) Rabbit mAb (upper) and α-Actinin (D6F6) XP® Rabbit mAb #6487 (lower). As expected, HCT 116 cells are negative for MLL3 expression.
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
MLL3 (D1S1V) Rabbit mAb recognizes endogenous levels of total MLL3 protein.Species Reactivity:
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ala2110 of human MLL3 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, mammals contain six Set1-related proteins: 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, which 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).
MLL3, also known as histone-lysine N-methyltransferase 2C (KMT2C), is a large 540 kDa protein that functions as part of the MLL3/COMPASS-like complex to activate gene expression by mediating mono-methylation of histone H3 lysine 4 at gene enhancers (7). Enhancer-specific H3 lysine 4 mono-methylation (H3K4me1) correlates with increased levels of chromatin interactions between gene enhancers and promoters, while loss of this modification results in a reduction of enhancer-promoter interactions (8). Furthermore, H3K4me1 facilitates recruitment of the Cohesin complex, which may function to promote the interactions between gene enhancers and promoters (8). MLL3 is found to be mutated or have altered expression in a number of different cancers (9).
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