Reagent name or type
Tested concentration range
Considerations for performing SP3 in the described conditions
Detergents
0-20%
Detergents tested in the listed concentration range include SDS, Triton X-100, NP-40, Tween 20, deoxycholate, CHAPS, and RapiGest. We recommend keeping the total detergent concentration in the range of 0–10% (wt/vol or vol/vol depending on the detergents used)
Chaotropes
0-8 M
Chaotropes tested in the listed concentration range include urea (up to 8 M), guanidinium hydrochloride, and isothiocyanate (up to4 M). High concentrations of chaotropes in the presence of the solvent used for binding can disrupt the interactions between SP3 beads and the proteins. Therefore, we recommend testing SP3 with your desired lysis solution formulation before further application
Salts
0-1 M
A wide range of salts have been tested, and we recommend keeping the final salt concentration in the lysate <1 M when using solvents for binding. High salt concentrations in the presence of binding solvent can disrupt the ability of proteins to efficiently localize on the SP3 bead surface. The critical concentration is going to depend on the identity and properties of the salt in question
Solvents
0-50%
Solvents tested in the listed concentration range include acetonitrile, acetone, isopropanol, ethanol, trifluoroethanol, and xylene. We recommend keeping the final solvent concentration in the lysate before binding <25% (vol/vol). If high solvent concentrations are present, the amount of ethanol added during SP3 can be scaled to achieve the desired final solvent concentration (e.g., 50% (vol/vol) final)
Hughes, C.S., Moggridge, S., Müller, T. et al. Single-pot, solid-phase-enhanced sample preparation for proteomics experiments. Nat Protoc 14, 68–85 (2019). https://doi.org/10.1038/s41596-018-0082-x