Cyclodextrin Screening Kit

General Information
The AthenaES Cyclodextrin Screening Kit is intended for identification of the best cyclodextrin for use in refolding proteins in a rapid screening format. Cyclodextrins have been used in protein refolding as a means of removing detergent from partially refolded protein solutions. This allows the protein to complete the refolding process. Since no universal protein refolding condition has been identified, the best conditions for a given protein must be determined empirically. The Cyclodextrin Screening Kit helps to simplify the process by providing three of the most commonly used cyclodextrins for protein refolding.


Cyclodextrin Screening Kit Components
10mL 100mM β-cyclodextrin
10mL 100mM methyl-β-cyclodextrin
10mL 100mM α-cyclodextrin
Applications Manual


Instructions
*Cyclodextrins are typically used in a two-step protein refolding procedure. In the first step the protein is partially refolded in the presence of detergent. It is recommend that a suitable detergent be identified or that an experimental design which screens both the detergent and the cyclodextrin simultaneously be used. The following algorithm is suggested for identifying a suitable cyclodextrin:

1.	Perform a preliminary screen of the physical-chemical parameters which are known to affect protein refolding. Parameters to test include pH, ionic strength, excipitents (i.e., detergents, polyols, chaotropic agents) redox state, temperature, and protein concentration. Refolding can be done by dilution, dialysis, or immobilization on a resin. Several commercial kits are available, such as Athena's Protein Refolding Kit, (cat #0600), which simplify the screening process by providing the pre-mixed buffers along with straightforward statistical analyses of the results. A detailed method can be found in the Athena's Protein Refolding Applications Manual.
2.	Identify a suitable detergent. Several different detergents should be tested to determine the best one for the given target protein. Optimization of the detergent concentration is not needed at this stage, but should be determined once the cyclodextrin has been selected.
3.	Perform the cyclodextrin screen. Perform the first step in the refolding process by employing the buffer composition and detergent identified in steps 1 and 2. The following protocol is recommended as it is one of the simplest to perform. a. Prepare three reactions of the refolding buffer wit the selected detergent and add the denatured protein to each solution slowly while mixing gently to give a final protein concentration of 50µg/ml. b. Incubate for 1h on ice. c. Add each cyclodextrin to a final concentration of 5mM, one cyclodextrin per reaction. Mix gently. d. Incubate for 1-24h. e. Assay the solution for refolded protein (by activity or physical means).
4.	Once the cyclodextrin has been selected, the optimum concentration of the detergent and cyclodextrin should be determined. The process can then be adopted for an alternate refolding scheme such as by dialysis, diafiltration, or chromatography.