Description

Nori Mouse MyBPC3 ELISA Kit Summary

Alternative names for MyBPC3: myosin-binding protein C, cardiac-type (MyBPC3 or cMyBP-C)

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: 

The myosin-binding protein C, cardiac-type (MyBPC3 or cMyBP-C) is a protein that in humans is encoded by the MYBPC3 gene.^[1] This isoform is expressed exclusively in heart muscle during human and mouse development,^[2] and is distinct from those expressed in slow skeletal muscle (MYBPC1) and fast skeletal muscle (MYBPC2). cMyBP-C is a 140.5 kDa protein composed of 1273 amino acids.^[3] cMyBP-C is a myosin-associated protein that binds at 43 nm intervals along the myosin thick filament backbone, stretching for 200 nm on either side of the M-line within the crossbridge-bearing zone (C-region) of the A band in striated muscle. The approximate stoichiometry of cMyBP-C along the thick filament is 1 per 9-10 myosin molecules, or 37 cMyBP-C molecules per thick filament. In addition to myosin, cMyBP-C also binds titin and actin.^[4] The cMyBP-C isoform expressed in cardiac muscle differs from those expressed in slow and fast skeletal muscle (MYBPC1 and MYBPC2, respectively) by three features: (1) an additional immunoglobulin (Ig)-like domain on the N-terminus, (2) a linker region between the second and third Ig domains, and (3) an additional loop in the sixth Ig domain.^[5] cMyBP-C appears necessary for normal order, filament length and lattice spacing within the structure of the sarcomere.^[6] cMyBP-C is not essential for sarcomere formation during embryogenesis, but is crucial for sarcomere organization and maintenance of normal cardiac function. Absence of cMyBP-C results in severe cardiac hypertrophy, increased heart-weight-to-body-weight-ratios, enlargement of ventricles, increased myofilament Ca2+ sensitivity and depressed diastolic and systolic function.^[7] Histologically, MyBPC3-targeted knock-out hearts display structural rearrangements with cardiac myocyte disarray and increased interstitial fibrosis similar to patients with hypertrophic cardiomyopathy, without obvious alterations in shape or size of single cardiac myocytes, a loss of lateral alignment of adjacent myofibrils with their Z-lines misaligned.^[7]

References

1. Gautel M, et al. (1995). The EMBO Journal. 14 (9): 1952–60.
2. Fougerousse F, et al. (1998). Circulation Research. 82 (1): 130–3.
3. Carrier L, et al. (1997). Circulation Research. 80 (3): 427–34.
4. Freiburg A, et al. (1996). European Journal of Biochemistry / FEBS. 235 (1–2): 317–23.
5. Winegrad S (1999). Circulation Research. 84 (10): 1117–26.
6. Colson BA, et al. (2007). Journal of Molecular Biology. 367 (1): 36–41.
7. Harris SP, et al. (2002). Circulation Research. 90 (5): 594–601.

Product Citations