Nori Rabbit Myostatin (GDF-8) ELISA Kit Summary

Alternative names for myostatin: GDF-8, growth differentiation factor 8, MSTN

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: 

Myostatin (also known as growth differentiation factor 8, abbreviated GDF-8) is a myokine, a protein produced and released by myocytes that acts on muscle cells’ autocrine function to inhibit myogenesis: muscle cell growth and differentiation. In humans it is encoded by the MSTN gene.^[1] Myostatin is a secreted growth differentiation factor that is a member of the TGF beta protein family.^[2] Animals either lacking myostatin or treated with substances that block the activity of myostatin have significantly more muscle mass. Furthermore, individuals who have mutations in both copies of the myostatin gene have significantly more muscle mass and are stronger than normal. Naturally occurring deficiencies of myostatin of various sorts have been identified in some breeds of cattle,^[3] sheep,^[4] whippets,^[5] and humans. In each case the result is a dramatic increase in muscle mass. Human myostatin consists of two identical subunits, each consisting of 109 amino acid residues.^[6] The protein is inactive until a protease cleaves the NH2-terminal, or “pro-domain” portion of the molecule, resulting in the active COOH-terminal dimer. Myostatin binds to the activin type II receptor, resulting in a recruitment of either coreceptor Alk-3 or Alk-4. This coreceptor then initiates a cell signaling cascade in the muscle, which includes the activation of transcription factors in the SMAD family-SMAD2 and SMAD3. Myostatin also inhibits Akt, a kinase that is sufficient to cause muscle hypertrophy, in part through the activation of protein synthesis. However, Akt is not responsible for all of the observed muscle hyperthrophic effects which are mediated by myostatin inhibition^[7] Thus myostatin acts in two ways: by inhibiting muscle differentiation, and by inhibiting Akt-induced protein synthesis.

References

1. Gonzalez-Cadavid NF, et al. (1998). PNAS. 95(25): 14938–43.
2. Carnac G, et al. (2006). Mini Reviews in Medicinal Chemistry. 6(7): 765–70.
3. Kambadur R, et al. (1997). Genome Research. 7(9): 910–16.
4. Clop A, et al. (2006). Nature Genetics. 38(7): 813–18.
5. Mosher DS, et al. (2007). PLoS Genetics. 3(5): e79. PMC 1877876.
6. Ge G, et al. (2006). Birth Defects Research. Part C, Embryo Today. 78(1): 47–68.
7. Sartori R, et al. (2014). Trends in Endocrinology and Metabolism. 25(9): 464–71.