GraDeR has been successfully applied to a number of different membrane protein complexes from different taxa, for which vastly different pufification protocols were used. So far, we have not seen or been reported any issues with any protein or protein complexes – On the contrary, GraDeR seems to work quite universally. Thus, the chance that GraDeR works well with your protein or protein complex. We kindly invite you to review and share your own experiences in our Forum.
GraDeR readily works with any solubilization detergent and buffer conditions we have tested so far and can be simply added at any stage of your purification protocol: GraDeR has been shown to stabilize membrane protein complexes while keeping them functionally active in all case we tested. Thus, the only step that needs to be optimized is the gradient centrifugation step: We recommend running conditions where the membrane protein complexes have migrated to approximately one third of the tube length away from the bottom of the centrifugation tube in order to ensure complete removal of detergent. The steepness, or even presence of an LMNG gradient can be optimized as well: stable complexes do, in many cases, not even require the presence of a detergent gradient.
For the complete removal of LMNG micelles, the high stability of micelles limits the applicability of standard protocols for detergent removal. We have tested several alternative approaches for detergent removal with no success, and have found gradient centrifugation a s a simple and reliable way to remove micelles under mild conditions. For an evaluation of alternatives, see the list below:
|Procedure to Remove Free LMNG||Issues|
|Size-exclusion chromatography (long, HPLC)||damage of fragile IMPs by loss of lipids and/orloss of membrane protein-bound LMNG through resin absorbance|
|Size-exclusion chromatography (short, gravimetric, or spin)||co-migration of LMNG micelles|
|Precipitation (by e.g. PEG6000)||co-precipitation of LMNG micelles|
|Sedimentation||potential co-sedimentation of LMNG micelles and loss of monodispersity (see precipitation)|
|Cyclodextrin||complexation difficult calibration|
|Density gradient centrifugation (GraDeR)||none|
The original publication of GRaDeR can be cited as follows: Hauer F, Gerle C, Fischer N, Oshima A, Shinzawa-Itoh K, Shimada S, Yokoyama K, Fujiyoshi Y, Stark H. GraDeR: Membrane Protein Complex Preparation for Single-Particle Cryo-EM. Structure. 2015 Sep 1;23(9):1769-75. doi: 10.1016/j.str.2015.06.029. Epub 2015 Aug 13.