Optimizing elution: Gentle Elution Buffer
Eluting your protein of interest is the final step in affinity chromatography before obtaining a pure, useful sample for further study. Common methods include elution using proteolytic elution, low pH elution, and denaturant elution. An alternative to these options is what’s known as Gentle Elution Buffer, or 3.6 M MgCl2 pH 6.6.
Originally marketed as an antibody elution buffer, Gentle Elution Buffer is useful for eluting other proteins in addition to antibodies from an affinity chromatography column. The high salt concentration of MgCl2 acts as a mild chaotrope by disrupting electrostatic interactions between the tag and the ligand without denaturing either. In the case of the CL7/Im7 system, this means that the non-covalent bond between CL7 and Im7 is broken while leaving both units intact. The near-neutral pH 6.6 of Gentle Elution Buffer avoids denaturing the tag, ligand, or the target protein itself. The CL7/Im7 bond is stable between pH 4.2-10 while the Im7 unit is stable from pH 3-10.
Common elution methods
The primary method of elution currently used in the CL7/Im7 system is proteolytic cleavage. By using highly pure and active proteases, you cleave the target protein from the column efficiently, resulting in natively folded, tag-free protein. However, some users may have concerns about steric hindrance or may desire to keep the tag intact. Guanidine will elute the protein with its tag but will denature the protein and the resin ligand in the process, requiring a refolding step. Guanidine is only used in the CL7/Im7 system to regenerate the Im7 column after proteolytic cleavage.
Gentle Elution Buffer
Gentle Elution Buffer combines the benefits of both methods. It elutes the protein with its tag without interfering with its structure or that of the Im7 ligand, so that the tag is intact for use in downstream assays if desired. Gentle Elution also solves concerns over steric hindrance in on-column protease cleavage. You can elute with MgCl2, dialyze MgCl2 out of the solution, cleave the tag off-column, and run the solution over the Im7 column which will trap the loose CL7, leaving only the target protein in the flow through fraction.
Gentle Elution Buffer is a useful alternative when proteases, low pH, or denaturants are undesirable in the elution process.