Dissection of Genetic Pathways in C. elegans - Caenorhabditis Elegans: Molecular Genetics and Development - page 136

Fig. 6 Tandem affinity purification (TAP) coupled with mass spectrometry (MS) analysis. The two
tags, GFP and S peptide domain, are fused to a gene product of interest. These two tags are separated by a
tobacco etch virus (TEV) protease cleavage site. The proteins associated with the gene product of interest
are isolated using the antibody against the first tag, GFP, which is then released by TEV protease cleavage.
S protein that binds to the S peptide domain is used for the secondary affinity purification. The associated
proteins are then separated from the gene product of interest and subject to MS analysis. (For color version
of this figure, the reader is referred to the web version of this topic.)
GFP that was removed by TEV protease digestion. A second round of affinity purifi-
cation using the second tag enriched the kinetochore complex components, which were
then subject to the MS analysis. MS analysis indicated that this two-step purification
process removed most of the nonspecific proteins that were present in the single-step
antibody-based immuoprecipitation that was also performed in parallel. The study
identified 10 kinetochore proteins, of which seven were previously uncharacterized.
4. Using Bioinformatics Tools
Bioinformatics is the analysis of biological systems, especially systems involving
genetic materials, using computer science, statistics, engineering, and information
theory. Bioinformatic tools have been widely applied in biological research, ranging
from sequence-based analysis, transcriptome analysis to computational proteomics
( Rhee et al., 2006 ). In C. elegans, bioinformatic tools in conjunction with functional
perturbation are effective in dissecting biological processes in some circumstances,
such as microRNA prediction ( Grad et al., 2003 ), gene identification by homology
search ( Berset et al., 2001; Chen and Greenwald, 2004 ), and cis-regulatory sequence