Western blot analysis of extracts from HeLa cells, untreated or staurosporine-treated, using Cleaved alpha-Fodrin (Asp1185) Antibody #2121 (left) and alpha-Fodrin Antibody (right).Learn more about how we get our images.
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.
Alpha-Fodrin Antibody detects endogenous levels of full length alpha-fodrin (240 kDa) and its large fragment (150 kDa) resulting from cleavage at aspartic acid 1185.
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Asp1185 of human alpha-fodrin. Antibodies are purified by protein A and peptide affinity chromatography.
Fodrin (also named nonerythroid spectrin) is a universally expressed membrane-associated cytoskeletal protein consisting of alpha- and beta-subunits (1). This protein is important for maintaining normal membrane structure and supporting cell surface protein function (1). Alpha-fodrin is one of the primary targets cleaved by caspases during apoptosis. The full length 240 kDa protein can be cleaved at several sites within its sequence by activated caspases to yield amino-terminal 150 kDa, carboxy-terminal 120 kDa and 35 kDa major products (2-5). Cleavage of alpha-fodrin leads to membrane malfunction and cell shrinkage.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
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