A Monoclonal Antibody That Inhibits Translation in Sf2l Cell Lysates Is Specific for Glyceraldehyde-3-Phosphate Dehydrogenase
A Monoclonal Antibody That Inhibits Translation in Sf2l Cell Lysates Is Specific for Glyceraldehyde-3-Phosphate Dehydrogenase
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DOI：
10.1002/arch.20271
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发表时间：
2008-11-01
影响因子：
2.2
通讯作者：
Stuart, Melissa K.
中科院分区：
文献类型：
--
作者：
Van Meter, Kipp E.;Stuart, Melissa K.
Monoclonal antibody (Mob) 8B7 was shown in a previous study to inhibit protein translation in lysates of Sf21 cells. The antibody was thought to he specific for a 60-kDa form of elongation factor-1 alpha (EF-1 alpha), primarily because the antigen immunoprecipitated by Mob 8B7 cross-reacted with Mob CBP-KK1, an antibody generated to EF-1 alpha from Trypanosoma brucei. The purpose of the current study was to investigate further the antigenic specificity of Mab 8B7. The concentration of the 60-kDa antigen relative to total cellular protein proved insufficient for its definitive identification. However, subcellular fractionation of Sf21 cells yielded air additional protein of 37 kDa in the cytosolic and microsomal fractions that was reactive with Mob 8B7. The 37-kDa protein could be easily visualized by colloidal Coomassie Blue G-250 staining as a series of pl 6.9-8.4 spots on two-dimensional gels. Excision of an abundant immunoreactive spot enabled identification of the protein as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) by matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) and protein database searching. Subsequent immunoblotting of purified rabbit skeletal muscle GAPDH with Mab 8B7 confirmed the antibody's specificity for GAPDH. Besides the pivotal role GAPDH plays in glycolysis, the enzyme has a number of noncanonical functions, including binding to mRNA and tRNA. The ability of Mob 8B7 to disrupt these lesser-known functions of GAPDH may account for the antibody's inhibitory effect on in vitro translation. Arch. Insect Biochem. Physiol. 69:107-117, 2008. (C) 2008 Wiley-Liss, Inc.
