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 and 50% glycerol. Store at –20°C. Do not aliquot the antibody.
E2A Antibody detects endogenous levels of total E2A protein.
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu475 of human E2A protein. Antibodies are purified by protein A and peptide affinity chromatography.
E2A is a member of the E-protein family of transcription factors, a subclass of basic helix-loop-helix (bHLH) proteins that bind specifically to E-box consensus sequences (1,2). Alternative splicing generates two E2A isoforms (E47 and E12) that are actively involved in B cell lineage commitment, B cell maturation, IgK V-J rearrangement, peripheral B cell development, and tumor suppression (3). E2A acts in cis during G1 to promote immunoglobulin gene diversification (4). Research studies have shown that chromosomal translocations involving the E2A gene result in the expression of multiple fusion proteins and are associated with many cases of pediatric acute lymphoblastic leukemia (5).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
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