An immunological method for detecting gene expression in yeast colonies.
AUTOR(ES)
Lyons, S
RESUMO
A method for detection of cloned, expressed genes in yeast colonies has been developed. The 70-kilodalton (kDa) mitochondrial outer membrane protein of yeast was used as a model protein. Transformation of a strain deficient in the gene for the 70-kDa protein was performed, and transformed colonies were detected with the antibody decoration technique. This technique is based upon gentle lysis of yeast colonies that have been grown on nitrocellulose filters such that the yeast proteins remain bound in discrete spots after lysis. The lysis is carried out by alkaline conditions in the presence of 2-mercaptoethanol and sodium dodecyl sulfate. After lysis, empty sites on the nitrocellulose filter are blocked to eliminate nonspecific binding of proteins by either 0.5% bovine serum albumin or 0.05% Tween 20. Decoration with antibody is visualized by using 125I-labeled protein A or peroxidase-conjugated second antibody. Antigens amounting to less than 0.1% of the total protein in the cell can be readily detected by the assay. The sensitivity of the assay enables detection of 1 positive colony per plate containing about 1000 colonies.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=392159Documentos Relacionados
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