Use of sodium dodecyl sulfate-polyacrylamide gel electrophoresis to resolve mRNA and its protein product in one gel.
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 [32P]H3PO4 and [35S]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 32P and not with 35S; 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.
Gao
W
W
Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey, Newark 07103, USA.
Goldman
E
E
D001345:Autoradiography / Q000379:methods; D012321:DNA-Directed RNA Polymerases / Q000096:biosynthesis* / Q000302:isolation & purification*; D003851:Deoxyribonucleases; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004790:Enzyme Induction / Q000187:drug effects; D007544:Isopropyl Thiogalactoside / Q000494:pharmacology; D008715:Methionine / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D010761:Phosphorus Radioisotopes; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000302:isolation & purification* / Q000378:metabolism; D015203:Reproducibility of Results; D012293:Rifampin / Q000494:pharmacology; D012967:Sodium Dodecyl Sulfate; D013462:Sulfur Radioisotopes; D014764:Viral Proteins
D010710:Phosphates; D010761:Phosphorus Radioisotopes; D012333:RNA, Messenger; D013462:Sulfur Radioisotopes; D014764:Viral Proteins; D007544:Isopropyl Thiogalactoside; D012967:Sodium Dodecyl Sulfate; D008715:Methionine; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases; D003851:Deoxyribonucleases; D012293:Rifampin