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.
Ero1-Lα Antibody detects endogenous levels of total Ero1-Lα protein.
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu218 of human Ero1-Lα. Antibodies are purified by protein A and peptide affinity chromatography.
Secretory proteins translocate into the endoplasmic reticulum (ER) after their synthesis where they are post-translationally modified and properly folded. To reach their native conformation, many secretory proteins require the formation of intra- or inter-molecular disulfide bonds (1). This process is called oxidative protein folding. Several oxidoreductases of the protein disulfide isomerase (PDI) family essential for disulfide formation and isomerization are localized to the ER (2). Studies have found that the ER-residing protein endoplasmic oxidoreductin-1 (Ero1) provides the oxidizing potential to the ER in Saccharomyces cerevisiae (3). In vitro experiments demonstrated that Ero1 is oxidized by molecular oxygen in a FAD-dependent manner and the oxidized Ero1 in turn serves as an oxidant for PDI (4). Two human homologs of Ero1, Ero1-like (Ero1-Lα and β) have been identified (2,5). Ero1-Lα is an ER membrane-associated N-glycoprotein that promotes oxidative protein folding and has been shown to be expressed in several cell lines and tissues (2).
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
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