Elimination of nonspecific bands in non-radioactive electrophoretic mobility shift assays using the digoxigenin system.
Anal Biochem
; 465: 70-2, 2014 Nov 15.
Article
em En
| MEDLINE
| ID: mdl-25004462
ABSTRACT
In the course of detecting nuclear transcription factors by electrophoretic mobility shift assay using digoxigenin (DIG)-labeled probes, we encountered a problem with a considerable nonspecific shift band in negative control lanes from which protein extracts were omitted. This nonspecific shift band can interfere with the detection of the desired target protein. Purification of the DIG-labeled probes by removing unincorporated DIG-labeled nucleotides did not resolve the problem. However, the introduction of an additional step of heating at 95 °C for 5 min and subsequent reannealing after DIG-labeled probe synthesis eliminated these nonspecific shift bands and allowed accurate analysis of the target protein.
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Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
DNA
/
NF-kappa B
/
Elementos de Resposta
/
Ensaio de Desvio de Mobilidade Eletroforética
/
Digoxigenina
Limite:
Humans
Idioma:
En
Ano de publicação:
2014
Tipo de documento:
Article