Assay Conditions

The single most important factor in these assays is that the apoprotein be fully reduced prior to addition of the bilin substrate. This is accomplished by using freshly purified apoprotein, adding DTT to 10 mM, and incubating this mixture for 30 minutes at room temperature (22). The DTT should be removed by gel filtration prior to addition of bilin. It has been observed that bilins will react with DTT when this compound is present at high concentrations (W.M. Schluchter and A.N. Glazer, unpublished results) (20). Generally, apophycobiliproteins are very stable when present in 5 to 50 mM Na-

Figure 4. HPLC separation of phycobiliproteins present in the phycobilisomes purified from Synechocystis sp. PCC 6803. Phy-cobilisomes from sucrose gradients were dialyzed extensively against 5 mM Na-phosphate, pH 7.0, prior to injection on a C4 reverse-phase column (see text for details). The elution of polypeptides was monitored at 280 nm (upper panel) and 680 nm (lower panel). The a-AP subunit is poorly resolved from the p-PC subunit.

Figure 4. HPLC separation of phycobiliproteins present in the phycobilisomes purified from Synechocystis sp. PCC 6803. Phy-cobilisomes from sucrose gradients were dialyzed extensively against 5 mM Na-phosphate, pH 7.0, prior to injection on a C4 reverse-phase column (see text for details). The elution of polypeptides was monitored at 280 nm (upper panel) and 680 nm (lower panel). The a-AP subunit is poorly resolved from the p-PC subunit.

phosphate, pH 7.0, or 10 to 50 mM Tris-HCl, pH 8.0, 75 mM NaCl, and therefore these conditions have been used in non-enzymatic assays.

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