Western blot analysis of extracts from various cell lines using Regucalcin/SMP30 Antibody (upper) and GAPDH (D16H11) XP® Rabbit mAb #5174 (lower). Expression levels of regucalcin/SMP30 among cell lines are consistent with expectations based on publicly available bioinformatic databases.
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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.
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
Regucalcin/SMP30 Antibody recognizes endogenous levels of total regucalcin/SMP30 protein.Species Reactivity:
Human, Mouse, Rat, Monkey
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to residues near the carboxy terminus of human regucalcin/SMP30 protein. Antibodies are purified by peptide affinity chromatography.
Regucalcin is a highly conserved, multifunctional protein encoded by the RGN gene on the X chromosome, generally recognized as a calcium (Ca2+) binding protein involved in the maintenance of intracellular Ca2+ homeostasis (1). The protein was independently identified and termed Senescence Marker Protein-30 (SMP30), following the observation in a rat aging model that expression of the protein markedly declined with age in a hormone-independent manner (2). Regucalcin/SMP30 has since been implicated in the regulatory control of many fundamental biological processes (e.g., cell growth, apoptosis, cellular metabolism), largely through its role in regulating Ca2+ homeostasis and Ca2+-dependent signal transduction (3-5). Research studies also suggest that regucalcin/SMP30 may function as a tumor suppressor in some contexts (3,6), while serum levels of regulcalcin/SMP30 have also been proposed as potential biomarkers of liver and kidney dysfunction (7).
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