A new multiphasic buffer system for sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins and peptides with molecular masses 100,000-1000, and their detection with picomolar sensitivity.
A novel multiphasic buffer system for high resolution sodium dodecyl sulfate-polyacrylamide gel electrophoresis of dansylated and nondansylated proteins/peptides in the relative molecular mass (Mr) range of 100,000-1000 is described. The system, based on Jovin's theory of multiphasic zone electrophoresis, allows complete stacking and destacking of proteins/peptides within the above Mr range. The buffer system uses Bicine and sulfate as trailing and leading ion, respectively, and Bistris and Tris as counter ions in the stacking and separating phase, respectively. Through selection of two different counter ions--the characteristic feature of the present ionic system--the stacking limits of a multiphasic buffer system can be further widened, thus making it applicable to gel electrophoresis of a larger spectrum of rapidly migrating species, such as sodium dodecyl sulfate-proteins/peptides and nucleic acids, than has been possible previously. Highly sensitive detection methods for proteins as well as for polypeptides down to approximately Mr 1000 are described. Dansylated proteins/peptides were detected by their fluorescence either directly within the gel or following electroblotting into anion-exchange or polyvinylidene difluoride membranes. The latter procedure resulted in detection sensitivities of approximately 1 ng. Nondansylated proteins/peptides were either detected within the gel by colloidal Coomassie staining or by electroblotting into polyvinylidene difluoride membranes, followed by colloidal gold staining. Prior to both staining procedures the proteins/peptides were pretreated with glutardialdehyde in the presence of borate at near neutral pH values to generate protein/peptide polymers of poor solubility. For a given pH the efficiency of the latter procedure was significantly influenced by the nature of the buffer ion used in the fixation buffer. In contrast to conventional fixation procedures even small polypeptides (Mr 1000) were immobilized and approximately 15 ng and 0.75 ng could be detected after colloidal Coomassie and colloidal gold staining, respectively.
Wiltfang
J
J
Max-Planck-Institut für experimentelle Medizin, Forschungsstelle Neurochemie, Göttingen.
Arold
N
N
Neuhoff
V
V
D002021:Buffers; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D003619:Dansyl Compounds; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005976:Glutaral; D005998:Glycine / Q000031:analogs & derivatives; D006046:Gold; D008970:Molecular Weight; D010455:Peptides / Q000032:analysis* / Q000302:isolation & purification; D011506:Proteins / Q000032:analysis* / Q000302:isolation & purification; D012394:Rosaniline Dyes; D012967:Sodium Dodecyl Sulfate; D013194:Staining and Labeling; D013431:Sulfates; D014325:Tromethamine / Q000031:analogs & derivatives
D002021:Buffers; D003619:Dansyl Compounds; D010455:Peptides; D011506:Proteins; D012394:Rosaniline Dyes; D013431:Sulfates; D014325:Tromethamine; C027494:N,N-bis(2-hydroxyethyl)glycine; D012967:Sodium Dodecyl Sulfate; D006046:Gold; C004692:coomassie Brilliant Blue; C026272:Bistris; C029038:dansyl chloride; D005976:Glutaral; D005998:Glycine