An estimation of the sensitivity of in vitro translation using two-dimensional gel analysis.
Poly (A+ mRNA species, isolated from 100-day-old rat brain, were analysed by in vitro translation and two-dimensional gel electrophoresis. The synthesis of selected protein species was compared to actin on the basis of [35S]methionine incorporation. The estimated molar abundance of translation products varied from abundant species at 0.78% of the total to several are species, detectable below the 0.02% level. If these synthesis rates reflect the abundance of particular mRNAs in the mixture, this sensitivity limit compares well with accepted values using differential cDNA screening techniques. This analysis provides evidence that in vitro translation methodology is able to detect rarer mRNA species than is usually expected--these include similar abundance classes to library screening procedures.
Perrett
C W
CW
Department of Neuroscience, Institute of Psychiatry, London, UK.
Whatley
S A
SA
D000199:Actins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics; D000818:Animals; D002846:Chromatography, Affinity; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D005260:Female; D015723:Gene Library; D008297:Male; D008970:Molecular Weight; D009419:Nerve Tissue Proteins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D011061:Poly A / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D016548:Prosencephalon / Q000502:physiology*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains
D000199:Actins; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger; D011061:Poly A