High resolution KSCN/CsSCN equilibrium gradients effectively separate a population of density labeled proteins from unlabeled proteins.
The persistence of proteins in a number of biological systems has been analyzed by density labeling techniques; however, the utility of this approach has been severely hampered by poor resolution between density-labeled and unlabeled proteins on equilibrium gradients. A high resolution equilibrium salt gradient composed of KSCN/CsSCN has been developed to effectively separate density-labeled proteins (13C-15N-2H-substituted) from unlabeled proteins. The resolution of this system is approximately twofold greater than that previously achieved with cesium formate/guanidine hydrochloride equilibrium gradients which have been used in many recent protein density labeling studies. In order to examine the extent of cross-contamination between density-labeled and unlabeled proteins in a KSCN/CsSCN gradient system, density-labeled chick epidermal proteins were mixed with unlabeled Drosophila larval proteins and then separated on these equilibrium gradients. From individual gradient fractions proteins were recovered and fractionated on a sodium dodecyl sulfate-polyacrylamide gel, demonstrating the virtually complete separation between the two populations. The general utility of this system for protein stability studies is also demonstrated.
Phillips
W H
WH
Department of Biology, University of Virginia, Gilmer Hall, Charlottesville, 22901.
Peterson
C A
CA
Grainger
R M
RM
D000818:Animals; D002247:Carbon Isotopes; D002499:Centrifugation, Density Gradient / Q000379:methods; D002586:Cesium; D002645:Chickens; D003903:Deuterium; D004331:Drosophila melanogaster / Q000032:analysis; D004817:Epidermis / Q000032:analysis; D007202:Indicators and Reagents; D007553:Isotope Labeling; D007814:Larva; D009587:Nitrogen Isotopes; D011506:Proteins / Q000302:isolation & purification*; D012867:Skin / Q000032:analysis; D013861:Thiocyanates
D002247:Carbon Isotopes; D007202:Indicators and Reagents; D009587:Nitrogen Isotopes; D011506:Proteins; D013861:Thiocyanates; D002586:Cesium; C059872:cesium thiocyanate; D003903:Deuterium; C009941:potassium thiocyanate