Biology Reference
In-Depth Information
challenging to sort relevant hits from background and necessitates redundant strat-
egies. New technical developments, including labeling-based and label-free meth-
ods, may also aid in this effort. In practical terms, the challenge in analyzing protein
mixtures by mass spectrometry lies primarily in developing a good working rela-
tionship with a mass spectrometry-focused laboratory or core facility.
Prioritization of potential interactors from lists generated by mass spectrometry of
immunoprecipiates/tandem affinity purifications for follow-up experiments is per-
haps the greatest challenge faced in biochemical analysis of protein complexes. In
this regard, it is helpful to think of the initial list of proteins identified as hits from a
primary screen, with a variety of secondary screens being necessary to separate the
wheat from the chaff. Redundant strategies and elimination of common contami-
nants are important means for filtering such lists. In addition, the extensive genomic
resources available in C. elegans that are archived on Wormbase provide an invalu-
able tool. Finally, the ability to rapidly test gene function is perhaps the most
important, as it motivates in-depth analysis to validate the initially observed putative
physical interaction.
IX. Summary
Although genetic and cell biological analysis continue to be central to elucidating
gene function in C. elegans, identifying protein-protein interactions is increasingly
being employed to develop comprehensive understanding of cellular pathways. In
this chapter, we discussed biochemical methods and outlined protocols currently
used to isolate protein complexes in C. elegans.
X. Solutions and Media
A. Worm Reagents
NGM agarose plates (100 mm and 60 mm)
3 g NaCl
25 g agarose
2.5 g peptone
975 mL ddH
2
O
Autoclave 35 min and place in 55
C water bath.
When cooled sterilely add:
1 mL cholesterol (5 mg/mL in EtOH)
1 mL 1 M CaCl
2
1 mL 1 M MgSO
4
25 mL 1 M KH
2
PO
4
(pH 6.0)
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