PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of neuroscibullNeuroscience Bulletin
 
Neurosci Bull. 2009 April; 25(2): 54–60.
Published online 2009 March 24. doi:  10.1007/s12264-009-1230-7
PMCID: PMC5552581

Language: English | Chinese

Preparation and application of rat myostatin antiserum

大鼠myostatin蛋白的抗体制备及其应用

Abstract

Objective

To prepare and identify a polyclonal antibody against rat myostatin and investigate myostatin expression in the rat atrophic gastrocnemius muscle after tibial nerve crush.

Methods

The purified fusion protein was used as antigen to immunize rabbits for the preparation of polyclonal antibody. The polyclonal antibody of the protein was measured by enzyme linked immunosorbent assay (ELISA), western-blot and immunochemistry. Myostatin protein expression levels in normal and atrophic gastrocnemius muscle were detected by western-blot and immunochemistry assays.

Results

The GST-myostatin had a purity of 96% and possessed high titer and specificity. The level of myostatin in gastrocnemius muscle significantly increased one week after tibial nerve crush, reached the peak on day 14, and then returned to normal level on day 28.

Conclusion

We have successfully made antiserum of rat myostatin and found that the expression level of myostatin protein in the gastrocnemius after tibial nerve crush-induced atrophy was time-dependent. This study provides an experimental basis to clarify the possible role of myostatin during skeletal muscle atrophy.

Keywords: myostatin, antibody, tibial nerve crush, western blot, immunohistochemistry

摘要

目的

采用GST-myostatin融合蛋白进行多克隆抗体的制备、 鉴定并进一步阐明神经损伤后腓肠肌肌캮缩过程中myostatin蛋白表达变化。

方法

将纯化好的GST-myostatin融合蛋白免疫家췃, 获得抗血清, 经Western blot, 免疫荧光组织化学及ELISA检测抗体特异性及效价; 建立大鼠胫神经볐伤模型, 采用western blot及免疫组织化学法检测胫神经볐伤后不同时间段腓肠肌中myostatin蛋白表达变化。

结果

得到较高纯度的GST-myostatin融合蛋白, 获得췃源的抗myostatin血清, 经western blot、 免疫组织化学及ELISA实验证实所得抗体具有较高的特异性及效价; 证实在胫神经볐伤后, 腓肠肌myostatin蛋白水平迅速上升, 在第14天达到高峰, 随后逐渐下降, 至第28天降至接近正常水平。

结论

成功制备了췃抗大鼠myostatin抗血清, 进一步检测表明了胫神经损伤后腓肠肌中myostatin蛋白的表达具有时间依赖性。 本研究为阐明myostatin在骨骼肌캮缩过程中的作用提供了实验依据。

关键词: myostatin, 抗体, 胫神经볐伤, western blot, 免疫组织化学

References

[1] McPherron A.C., Lee S.J. Double muscling in cattle due to mutations in the myostatin gene. Proc Natl Acad Sci USA. 1997;94(23):12457–12461. doi: 10.1073/pnas.94.23.12457. [PubMed] [Cross Ref]
[2] McPherron A.C., Lawker A.M., Lee S.J. Regulation of skeletal muscle mass in mice by a new TGF-β superfamily member. Nature. 1997;387:87–90. doi: 10.1038/387083a0. [PubMed] [Cross Ref]
[3] Grobet L., Martin L.J., Poncelet D., Pirottin D., Brouwers B., Riquet J., et al. A deletion in the bovine myostatin gene causes the double-muscled phenotype in cattle. Nat Genet. 1997;17:71–74. doi: 10.1038/ng0997-71. [PubMed] [Cross Ref]
[4] Grobet L., Poncelet D., Royo L.J., Brouwers B., Pirottin D., Michaux C., et al. Molecular definition of an allelic series of mutations disrupting the myostatin function and causing double-muscling in cattle. Mamm Genome. 1998;9:210–213. doi: 10.1007/s003359900727. [PubMed] [Cross Ref]
[5] Clop A., Marcq F., Takeda H., Pirottin D., Tordoir X., Bibé B., et al. A mutation creating a potential illegitimate microRNA target site in the myostatin gene affects muscularity in sheep. Nat Genet. 2006;38:813–818. doi: 10.1038/ng1810. [PubMed] [Cross Ref]
[6] Mosher D.S., Quignon P., Bustamante C.D., Sutter N.B., Mellersh C.S., Parker H.G., et al. A Mutation in the myostatin gene increases muscle mass and enhances racing performance in heterozygote dogs. PLoS Genet. 2007;3:e79. doi: 10.1371/journal.pgen.0030079. [PMC free article] [PubMed] [Cross Ref]
[7] Schuelke M., Wagner K.R., Stolz L.E., Hübner C., Riebel T., Kömen W., et al. Myostatin mutation associated with gross muscle hypertrophy in a child. New Engl J Med. 2004;350:2682–2688. doi: 10.1056/NEJMoa040933. [PubMed] [Cross Ref]
[8] Rodgers B.D., Roalson E.H., Weber G.M., Roberts S.B., Goetz F.W. A proposed nomenclature consensus for the myostatin gene family. Am J Physiol: Endocrinol Metab. 2007;292:E371–E372. doi: 10.1152/ajpendo.00395.2006. [PubMed] [Cross Ref]
[9] Maccatrozzo L., Bargelloni L., Radaelli G., Mascarello F., Patarnello T. Characterization of the myostatin gene in the gilthead seabream, Sparus aurata: sequence, genomic structure, and expression pattern. Mar Biotechnol. 2001;3:224–230. doi: 10.1007/s101260000064. [PubMed] [Cross Ref]
[10] Maccatrozzo L., Bargelloni L., Cardazzo B., Rizzo G., Patarnello T. A novel second myostatin gene is present in teleost fish. FEBS Lett. 2001;509:36–40. doi: 10.1016/S0014-5793(01)03124-6. [PubMed] [Cross Ref]
[11] Wehling M., Cai B., Tidball J.G. Modulation of myostatin expression during modified muscle use. FASEB J. 2000;14:103–110. [PubMed]
[12] Zhang D.L., Liu M., Ding F., Gu X. Expression of myostatin RNA transcript and protein in gastrocnemius muscle of rats after sciatic nerve resection. J Muscle Res Cell Mot. 2006;27:37–44. doi: 10.1007/s10974-005-9050-5. [PubMed] [Cross Ref]
[13] Liu M., Zhang D.L., Shao C.X., Liu J., Ding F., Gu X. Expression pattern of myostatin in gastrocnemius muscle of rats after sciatic nerve crush injury. Muscle Nerve. 2007;35:649–656. doi: 10.1002/mus.20749. [PubMed] [Cross Ref]
[14] Hu Y.X., Guo J.Y., Shen L., Chen Y., Zhang Z.C., Zhang Y.L. Get effective polyclonal antisera in one month. Cell Res. 2002;12:157–160. doi: 10.1038/sj.cr.7290122. [PubMed] [Cross Ref]
[15] Roberts S.B., McCauley L.A.R., Devlin R.H., Goetz F.W. Transgenic salmon overexpressing growth hormone exhibit decreased myostatin transcript and protein expression. J Exp Biol. 2004;207:3741–3748. doi: 10.1242/jeb.01210. [PubMed] [Cross Ref]
[16] Jackman R.W., Kandarian S.C. The molecular basis of skeletal muscle atrophy. Am J Physiol Cell Physiol. 2004;287:834–843. doi: 10.1152/ajpcell.00579.2003. [PubMed] [Cross Ref]
[17] Thomas M., Langley B., Berry C., Sharma M., Kirk S., Bass J., et al. Myostatin, a negative regulator of muscle growth, functions by inhibiting myoblast proliferation. J Biol Chem. 2000;275:40235–40243. doi: 10.1074/jbc.M004356200. [PubMed] [Cross Ref]
[18] Carlson C.J., Booth F.W., Gordon S.E. Skeletal muscle myostatin mRNA expression is fiber-type specific and increases during hindlimb unloading. Am J Physiol. 1999;277:R601–R606. [PubMed]
[19] McMahon C.D., Popovic L., Oldham J.M., Jeanplong F., Smith H.K., Kambadur R., et al. Myostatin-deficient mice lose more skeletal muscle mass than wild-type controls during hindlimb suspension. Am J Physiol Endocrinol Metab. 2003;285:E82–E87. [PubMed]

Articles from Neuroscience Bulletin are provided here courtesy of Springer