Western blot analysis of extracts from Jurkat and U937 cells, using Calpain 1 Large Subunit (Mu-type) Antibody.
|REACTIVITY||H M R|
|MW (kDa)||75, 80|
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 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
Calpain 1 Large Subunit (Mu-type) Antibody detects endogenous levels of total calpain 1 (large subunit) protein. The antibody detects full-length calpain 1 as well as calpain 1 autoproteolytically cleaved at leucine 28.Species Reactivity:
Human, Mouse, RatSpecies predicted to react based on 100% sequence homology:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to the human sequence of calpain 1 (large subunit). Antibodies are purified by protein A and peptide affinity chromatography.
Calpain is a calcium-dependent thiol proteinase that is functionally active as a heterodimer composed of a small regulatory subunit and one of at least two large catalytic subunits (calpain 1 or calpain 2). In vitro, calpain 1 (mu-calpain) requires micromolar levels of calcium, while calpain 2 (M-calpain) requires millimolar levels of calcium for activation. The regulation of calpain in vivo is the subject of many current studies, which suggest that proteolytic activity is regulated post-transcriptionally by mechanisms such as calcium requirements, subcellular localization of the heterodimer, phosphorylation via the EGFR-Erk signaling cascade, endogenous inhibitors (calpastatin) and autoproteolytic cleavage (1). Calpastatin negatively regulates autoproteolytic cleavage of calpain 1 between Gly27 and Leu28 (2). Calpain influences cell migration by modifying rather than degrading its substrates responsible for cell adhesion and cytoskeletal arrangement. Control of calpain activity has caught the attention of drug development since limiting its activity could mute invasiveness of tumors or chronic inflammation (1).
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