Description

Nori Rabbit cTnT ELISA Kit Summary

Alternative names for cTnT: Cardiac troponin T, TNNT2

Standard Curve

Kit Components
1. Pre-coated 96-well Microplate
2. Biotinylated Detection Antibody
3. Streptavidin-HRP Conjugate
4. Lyophilized Standards
5. TMB One-Step Substrate
6. Stop Solution
7. 20 x PBS
8. Assay Buffer

Other Materials Required but not Provided:
1. Microplate Reader capable of measuring absorption at 450 nm
2. Log-log graph paper or computer and software for ELISA data analysis
3. Precision pipettes (1-1000 µl)
4. Multi-channel pipettes (300 µl)
5. Distilled or deionized water

Protocol Outline
1. Prepare all reagents, samples and standards as instructed in the datasheet.
2. Add 100 µl of Standard or samples to each well and incubate 1 h at RT.
3. Add 100 µl of Working Detection Antibody to each well and incubate 1 h at RT.
4. Add 100 µl of Working Streptavidin-HRP to each well and incubate 20 min at RT.
5. Add 100 µl of Substrate to each well and incubate 5-30 min at RT.
6. Add 50 µl of Stop Solution to each well and read at 450 nm immediately.

Background:

Cardiac Troponin T (TnT), is a protein which in humans is encoded by the TNNT2 gene.^[1] Cardiac TnT is the tropomyosin-binding subunit of the troponin complex, which is located on the thin filament of striated muscles and regulates muscle contraction in response to alterations in intracellular calcium ion concentration. Cardiac TnT is a 35.9 kDa protein composed of 298 amino acids.^[2] Cardiac TnT is the largest of the three troponin subunits (cTnT, troponin I (TnI), troponin C (TnC)) on the actin thin filament of cardiac muscle. The structure of TnT is asymmetric; the globular C-terminal domain interacts with tropomyosin (Tm), TnI and TnC, and the N-terminal tether which strongly binds Tm. The N-terminal region of TnT is alternatively spliced, accounting for multiple isoforms observed in cardiac muscle.^[3] As part of the Troponin complex, the function of cTnT is to regulate muscle contraction. The N-terminal region of TnT that strongly binds actin most likely moves with Tm and actin during strong myosin crossbridge binding and force generation. This region is likely involved in the transduction of cooperativity down the thin filament. The C-terminal region of TnT constitutes part of the globular troponin complex domain and participates in employing the calcium sensitivity of strong myosin crossbridge binding to the thin filament. Mutations in this gene have been associated with familial hypertrophic cardiomyopathy as well as with restrictive^[4] and dilated cardiomyopathy. Transcripts for this gene undergo alternative splicing that results in many tissue-specific isoforms, however, the full-length nature of some of these variants has not yet been determined. Mutations of this gene may be associated with mild or absent hypertrophy and predominant restrictive disease, with a high risk of sudden cardiac death.^[4] Advancement to dilated cardiomyopathy may be more rapid in patients with TNNT2 mutations than in those with myosin heavy chain mutations.^[5]

References

1. Townsend PJ, et al. (1994). Genomics 21 (2): 311–6.
2. Zong, N. C. et al. (2013). Circulation Research 113 (9): 1043–53.
3. Anderson PA, et al (1991). Circulation Research 69 (5).
4. Revera M, et al. (2007). Cardiovascular Journal of Africa 18 (3): 146–53.
5. Fujino N, et al. (2002). The American Journal of Cardiology 89 (1): 29–33.

Product Citations