Additional Spin Column Methods

1 Competition Experiments of Inhibitor Mixture with Protein Taiget 

19A with respect to the corresponding reference peaks in Fig. 2.19B, C. Note that all spectra are normalized to the same intensity scale and were obtained using 10 pL of the mixture where the protease concentration was 20 pM in each sample. Reprinted from reference [13] with permission from John Wiley Sons. 

An important question that needs to be addressed in any screening study is the determination of whether or not the ligand is non-covalently bound to the active site of the target protein. A number of simple GPC spin column ESI-MS screening methods have been developed to answer this question. These methods include the use of mutated proteins where the active site has been modified, GPC spin column/ESI-MS coupled with NMR (GPC spin column/MS/NMR) and displacement of known binders. Titration experiments with molar excesses of ligand to protein (described below in Section 2.3.3.2.4) can also be used to determine whether single or multiple binding sites are available in the protein. 

1 Comparing Non-covalent Binding of Ligand to Mutated Proteins 

The GPC spin column/ESI-MS methodology with mutated CMV proteases was utilized to characterize the non-covalent binding site of ligand inhibitors [13]. The following illustration demonstrates the use of the GPC spin column screening technique to characterize non-covalent binding of TFMK to specific sites 

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While these methods provided efficient means for the purification of enzymatic products, they also proved to be a hurdle for high-throughput applications centrifugation steps, such as those required for spin columns, are difficult to automate. Sohd-phase purification systems usually require several reagent addition and washing steps, and therefore require more elaborate pipetting systems. 

Purify the derivatized dendrimer using gel filtration (size exclusion chromatography) on a desalting column or through use of ultrafiltration spin-tubes (for G-4 and above). For smaller dendrimers, the derivatives may be purified by repeated precipitation from a meth-anolic solution by addition of ethyl acetate, dioxane, or benzene. The SPDP-dendrimer may be dried by lyophilization (if in water or buffer) or by solvent evaporation in vacuo (if the precipitation method was used). 

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