Biomedical Engineering Reference
In-Depth Information
The presence of specific proteins can then be identified by western blot
analysis.
Southwestern blot
A southwestern blot is one method that is sometimes used to iden-
tify DNA-binding proteins, and characterize their binding specificity. Nu-
clear proteins, purified from nuclei isolated from homogenized cells are
resolved by SDS-PAGE, transferred to a membrane and probed with
oligonucleotide probes. The oligonucleotides that are used can corre-
spond to consensus DNA motifs recognized by defined transcription
factors, or their mutant counterparts. This method works for some, but
not many, DNA binding proteins because the DNA binding sites often
depend on three-dimensional folding patterns that are often disrupted
during protein processing.
Yeast two-hybrid screen
The yeast two-hybrid system is a powerful system for identifying inter-
acting transcriptional activators. It was described in detail in Chapter 1.
H.
Silencing gene expression
Introduction
A number of approaches are used to determine the function of a gene.
cDNA encoding a gene can be expressed in cells in wild-type and mutant
forms, using site-directed mutagenesis. However, it is often desirable to
produce a functional knockout of a gene in order to determine the effect
that the failure to express a particular gene has cellular function. Knock-
outs can be accomplished in several different ways. First, a gene can be
functionally deleted in embryonic stem cells by homologous recombi-
nation, and the deletion introduced into the germline in animals. This is
discussed in Chapter 6. Methods have also been developed to function-
ally inactivate a gene's expression using knockdown or gene silencing
approaches. These methods can be used in certain organisms and in
some, but not all cell types. These methods are briefly described below.
Antisense RNA
Antisense RNA is a method that attempts to block translation of mRNA
by introduction of a corresponding minus strand nucleic acid into the
cell. The minus strand nucleic acid can be introduced by transfection
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