Purification of 70S ribosomes.
Here we describe the further purification of prokaryotic ribosomal particles obtained after the centrifugation of a crude cell lysate through a sucrose cushion. In this final purification step, a fraction containing ribosomes, ribosomal subunits, and polysomes is centrifuged through a 7%-30% (w/w) linear sucrose gradient to isolate tight couple 70S ribosomes, as well as dissociated 30S and 50S subunits. The tight couples fraction, or translationally active ribosome fraction, is composed of intact vacant ribosomes that can be used in cell-free translation systems.
Department of Biology and Center for the Study of Biological Complexity, Virginia Commonwealth University, Richmond, Virginia 23284;
Maria C
MC
Rivera
Primary Pharmacology Group, Pfizer Global Research and Development, Groton, Connecticut 06340;
Bruce
B
Maguire
Department of Molecular, Cell and Developmental Biology and Department of Human Genetics, University of California, Los Angeles, California 90095.
James A
JA
Lake
D002499:Centrifugation, Density Gradient / Q000379:methods*; D045424:Complex Mixtures; D011387:Prokaryotic Cells / Q000737:chemistry; D012270:Ribosomes
D045424:Complex Mixtures