Western blot analysis of extracts from 293T cells, mock transfected (-) or transfected with a human cardiac Troponin T (isoform 1) construct (+), using Troponin T (Cardiac) Antibody.
Western blot analysis of extracts from human and rat heart and human skeletal muscle using Troponin T (Cardiac) Antibody.
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
Troponin T (Cardiac) Antibody detects endogenous levels of total cardiac Troponin T protein.Species Reactivity:
Human, RatSpecies predicted to react based on 100% sequence homology:
Polyclonal antibodies are produced by immunizing animals with a synthetic peptide corresponding to a region surrounding Pro69 of human cardiac Troponin T protein. Antibodies are purified by protein A and peptide affinity chromatography.
Troponin, working in conjunction with tropomyosin, functions as a molecular switch that regulates muscle contraction in response to changes in the intracellular Ca2+ concentration. Troponin consists of three subunits: the Ca2+-binding subunit troponin C (TnC), the tropomyosin-binding subunit troponin T (TnT), and the inhibitory subunit troponin I (TnI) (1). In response to β-adrenergic stimulation of the heart, Ser23 and Ser24 of TnI (cardiac) are phosphorylated by PKA and PKC. This phosphorylation stimulates a conformational change of the regulatory domain of TnC, reduces the association between TnI and TnC, and decreases myofilament Ca2+ sensitivity by reducing the Ca2+ binding affinity of TnC (1-3).
The tropomyosin binding subunit of the troponin complex TnT exists as different isoforms in slow skeletal muscle (ssTnT/TNNT1), fast skeletal muscle (fsTnT/TNNT3) and in cardiac muscle (cTnT/TNNT2). Each of these may also contain multiple alternatively spliced variants. Assays for measuring serum concentrations of cTnT, as well as cTnI, have been reported for analyzing cardiac injury.
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