To continue our detergent theme, here’s an affinity-based detergent removal method that is faster than the gel-assisted proteolysis but removes only the detergents, leaving the salts and chaotropic agents for you to deal with later. Unlike the gel-assisted method, it works for both proteins and peptides. The gel-assisted method is best suited for proteins, because the small, more soluble peptides are likely to elute out of the gel matrix during the washing.
I use Pierce detergent removal spin columns (0.125 mL format) in my lab, but there are other options available such as Bio-Beads or HyperD, each with its own pros and cons in the business of detergent removal. Pierce also sells a so-called HiPPR detergent removal resin (high protein and peptide recovery) for low-protein-concentration samples. The initial % detergent in such samples must also be low (ca. 1%).
The Pierce resin removes common ionic, nonionic, and zwitterionic detergents from protein and peptide solutions. This oligosaccharide-based affinity resin has a small hydrophobic cavity which creates a microenvironment for a detergent’s nonpolar moiety to enter and form an inclusion complex.
The workflow is simple: (1) centrifuge the column to remove storage buffer, (2) wash 3 times with your favorite buffer (pH 5-10), discarding the buffer each time, (3) add protein or peptide solution and let the resin do its magic for 2-5 min at room temperature, (4) centrifuge to collect your >95% detergent-free sample, i.e. don’t discard the flow-through this time!
Antharavally and co-workers from Thermo Scientific Pierce Protein Research published a study examining the detergent removal efficiency and protein recovery using their resin under several conditions (doi:10.1016/j.ab.2011.05.013). A table from this reference gives you some idea of the detergent concentrations removable with the Pierce resin.
Samples (0.1 ml containing 0.100 mg BSA + detergent at maximum concentration) were processed through 0.5 ml of Pierce detergent removal resin, and the residual detergent was measured as described in Materials and Methods. Protein concentration was determined by BCA protein assay (Pierce).