Use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis to resolve mRNA and its protein product in one gel

被引:4
作者
Gao, WW
Goldman, E
机构
[1] UNIV MED & DENT NEW JERSEY,NEW JERSEY MED SCH,DEPT MICROBIOL & MOL GENET,NEWARK,NJ 07103
[2] UNIV MED & DENT NEW JERSEY,GRAD SCH BIOMED SCI,DEPT MICROBIOL & MOL GENET,NEWARK,NJ 07103
关键词
codon usage in Escherichia coli; rare arginine codons; translational control;
D O I
10.1096/fasebj.11.13.9367350
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
We demonstrate that it is possible to simultaneously resolve both an mRNA and its protein product by electrophoresis in a single SDS-polyacrylamide gel by using double labeling with [P-32]H3PO4 and [S-35]methionine, and an elongated 5% stacking gel atop the 10% resolving gel, The mRNA is resolved in the 5% gel; the protein, as expected, resolves in the 10% gel. Using a T7 expression system, we show that putative mRNA bands in the 5% gel are: 1) labeled only with P-32 and not with S-35; 2) inducible with isopropylthiogalactopyranoside (needed to induce a T7 RNA polymerase gene under control of a lac promoter); 3) synthesized in the presence of rifampicin (T7 RNA polymerase is not inhibited by rifampicin); 4) degraded by base or RNase treatment; and 5) are largely resistant to DNase treatment, The mRNA bands were also evident in samples not treated with rifampicin, We used this technique to confirm previously published results that inhibition of expression by consecutive low-usage AGG arginine codons inserted near the 5' end of a test message in Escherichia coli is at the level of translation.
引用
收藏
页码:1153 / 1156
页数:4
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