The second step in a successful coprecipitation is generating whole-cell extracts that optimize the yield and activity of the proteins to be analyzed, using lysis buffer conditions that permit recognition of the proteins by the affinity matrix. The yield of total protein in a whole-cell extract is not always a reliable indicator of the relative yield and activity of specific proteins, so it is wise to verify both parameters at the onset of an experiment before proceeding with the coprecipitation. Yield and activity can be affected by a number of factors (see Chapter 10; see Harlow and Lane, 1988). Small variations in the relative amounts of salt and detergents in the lysis buffer can have large effects on yield and activity, as can the speed and efficiency of cell breakage. Both factors are particularly important for less soluble proteins that associate with macromolecular structures such as membranes or cytoskeleton. In addition, global inhibition of proteolysis through the inclusion of multiple classes of protease inhibitors may be essential.
Methods for preparing whole-cell extracts from yeast (units5.6-5.8), Escherichia coli (unit 6.2), insect cells (unit5.12), and mammalian cells (units 5.9 & 5.10) can be found elsewhere in this manual, and specifics will not be discussed here. In general, the lysis buffer conditions are not very different from the coprecipitation conditions. It is recommended that the investigator begin by comparing small-scale extract preparations that vary the amount of salt and nonionic detergent. As a starting point, a basic lysis buffer might contain the following components.
Basic components. Basic components include a buffering agent (such as 50 mM Tris Cl, pH 7.5), a small amount of nonionic detergent (such as 0.1% [v/v] Triton X-100), salt (such as 100 mM NaCl), a reducing agent (such as 1 mM DTT), and 10% (v/v) glycerol as stabilizer.
Protease inhibitors. Protease inhibitor cocktails are described here in unit5.8 and are also commercially available. A reasonable starting point would be to include 5 ^g/ml each of chymostatin, pepstatin A, leupeptin, and antipain, as well as 1 mM phenylmethysulfonyl fluoride and 1 mM benzamidine.
Detection of Protein-Protein Interactions by Coprecipitation
Chelating agents. EGTA (~15 mM) is commonly included to chelate divalent metal ions that are essential for metalloprotease activity. Because EGTA also inhibits other metal-dependent enzymes, it may be omitted, combined with the addition of a needed metal ion, and/or substituted with EDTA.
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