Preparation And Application Of Rat Myostatin Antibody

Objective To express the rat myostatin protein in Escherichia coli (BL21) and purify myostatin by electroeluting. 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 To make GST-Myostatin fusion protein in Escherichia coli (BL21) under the optimized induction of isopropyl-β-D-thiogalactopyranoside (IPTG). Western-blot analysis was performed using anti-GST antibody to confirm the fusion protein, which was then purified by electroeluting. The purified fusion protein was used as antigen to immunize rabbits for the preparation of polyclonal antibodies. The polyclonal antibody of the protein was measured by enzyme linked immunosorbent assay (ELISA), Western-blot and immunohistochemistry. Myostatin gene and protein expression levels in normal and atrophic gastrocnemius muscle were detected by RT-PCR,Western-blot and immunohistochemistry 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 possessed the GST-myostatin fusion protein. We have successfully made an antiserum of rat myostatin and found that the expression 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.