Mapping the Genotype–Phenotype Relationship in Cellular Signaling Networks: Building Bridges Over the Unknown - Systems Biology: Networks, Models, and Applications - page 147

coupled to a matrix column, proteins to be equally preparation.

and only those proteins considered. False negatives, because

that bind to it are retained Highly sensitive to even the complexes may not form due to

from an appropriate extract weakest of protein-protein a lack of appropriate protein

(that is ultimately eluted by, interactions. modifications, or due to the

e.g., a high-salt concentration), Enables the detection of both associated resin.

whereas all others pass the domains and critical Amount of extract applied is

through the affinity residues of a protein that are highly critical: too little of one

chromatography column or responsible for a specific kind of protein may cause

are readily washed off under interaction. interactions to go unnoticed,

low-salt conditions. Interactions that depend whereas too much may cause

upon multi-subunit minor ligand species to be

tethering can be detected. missed.

Protein probing Indicates which proteins in an Enables the unbiased testing Proteins encoded by the library [27,46]

expression library bind to a of multiple ligands against a may not fold or undergo

specific probe ligand. specific target protein, appropriate posttranslational

A labeled protein is used enabling, e.g., less abundant modifications in E. coli and/or

as a probe to screen an proteins to be equally on a nitrocellulose structure.

expression library, and considered.

Binding conditions are arbitrarily

interactions between an The protein probe may be imposed by the investigator, as

immobilized (library) manipulated in vitro to opposed to mimicking the native

protein and the labeled introduce a specific environment.

probe protein occur on an posttranslational modification

All possible combinations of

intermediary nitrocellulose that is essential for the protein-protein interactions

filter. The filter is ability of the probe to bind are assayed, including those

subsequently washed of to ligand(s) in the that might never occur in vivo.

nonbinding compounds library.

Continued