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
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.
BCL9 Antibody recognizes endogenous levels of total BCL9 protein. The antibody also cross-reacts with an unidentified protein of 21 kDa in some cell lines.
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding His138 of human BCL9 protein. Antibodies are purified by protein A and peptide affinity chromatography.
B-cell CLL/lymphoma 9 protein (BCL9) is a widely conserved adaptor protein that functions as a transcriptional co-activator in the canonical Wnt signaling pathway (1,2). BCL9 is a core component of a nuclear protein complex (BCL9, LEF/TCF, β-catenin and PYGO) that regulates the transcription of Wnt-dependent target genes (3). Research studies show that disrupting the interaction between BCL9 and β-catenin suppresses oncogenic Wnt signaling, suggesting a potential avenue for therapeutic intervention in Wnt-mediated cancers (4). BCL9 promotes association of PYGO with the tail of histone H3 that has been methylated at lysine 4 (H3K4me), suggesting a specific chromatin remodeling function for BCL9 in the Wnt signaling pathway (5). Research studies in colon epithelium and adenocarcinomas suggest that BCL9 is required to mediate Wnt-dependent stem cell behaviors, such as epithelial-mesenchymal transition (6). Crystallography studies revealed that BCL9 contains a β-catenin binding site that is distinct from the majority of known β-catenin binding partners, making it an attractive target for therapeutic drug development (7).
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