Effect of protein application mode and acrylamide concentration on the resolution of protein spots separated by two-dimensional gel electrophoresis.
Electrophoresis
; 18(11): 2085-90, 1997 Oct.
Article
em En
| MEDLINE
| ID: mdl-9420174
ABSTRACT
Two-dimensional gel electrophoresis separates large numbers of proteins in two steps on the basis of differences in their pIs and molecular masses. The separation is usually performed on immobilized pH gradient strips, followed by gradient polyacrylamide gels separating proteins with molecular masses between 5-200 kDa. For the first-dimensional separation the protein samples are usually applied near one end of the strip. Using total soluble protein extracts of the bacterium Haemophilus influenzae, we found that simultaneous sample application at both the basic and the acidic ends of the strip resulted in detection of more and stronger protein spots in comparison with sample application at one end only. Because many proteins of an organism have similar pI and Mr values, an overlapping of protein spots is frequently observed in the second-dimensional separation. The soluble protein fraction of H. influenzae was further separated on gels of constant acrylamide concentration between 7.5% and 15.0%. We found that for proteins of molecular mass within certain ranges, the gels of homogeneous acrylamide concentration provided more efficient spot separation than the gradient gels. The observed improvements in spot resolution may be useful in the characterization of proteins from other organisms or cell lines.
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Coleções:
01-internacional
Contexto em Saúde:
3_ND
/
4_TD
Base de dados:
MEDLINE
Assunto principal:
Proteínas de Bactérias
/
Acrilamidas
/
Eletroforese em Gel Bidimensional
Idioma:
En
Revista:
Electrophoresis
Ano de publicação:
1997
Tipo de documento:
Article