OptiPrep® is a 60 % (w/v) solution of iodixanol in sterile water provided by Axis Shield or its distributor (Sigma-Aldrich, Greiner) as a ready-to-go solution for different gradient applications. My application using OptiPrep® was the isolation of lipid rafts from membranes of cultured primary brain cells. When using sucrose gradients, e.g. for isolations of lipid rafts from Triton-X 100 soluble cell membrane fractions, it is always necessary to prepare fresh sucrose solutions on the day the gradient is established in order to avoid bacterial contamination and subsequent degradation of the gradient. To obtain consistent and reliable separation results when using a sucrose gradient, care has to be taken in precisely preparing the same percentages of sucrose solutions always in the same way. This can be very time consuming, especially since higher sucrose solutions are difficult to dissolve.
OptiPrep® has the advantage of being supplied as a ready-to-go sterile solution that can be taken out of the bottle with a sterile needle and syringe in the amount needed. OptiPrep® should be stored at room temperature, in the dark. (I kept it in the box in which it was shipped.) Since it is a non-sticky, water-based solution, OptiPrep® is easy to mix with common buffers (e.g. HEPES, or HEPES/NaCl) containing different protease inhibitors commonly used in protein biochemistry when isolating protein homogenates from cells or tissues. Since OptiPrep® is provided in water, it is necessary to use higher buffer concentrations than standard in order to obtain the desired final concentration. Different percentages of OptiPrep® can then be used to prepare either step (e.g. 10%, 20%, 30%) or continuous gradients (e.g. 10-30%). The gradient can be made isoosmotic at any density. Because OptiPrep® has low osmolality, it is necessary to include in the diluent an osmotic balancer (e.g. NaCl).
For the isolations of lipid raft, I prepared a step gradient. I first mixed the Triton-X 100 solubilized membrane lysate (in HEPES/NaCl) with 60% OptiPrep® to obtain a 45% solution. I then overlaid this protein-containing OptiPrep® solution (45%) with 35% OptiPrep® and 5% OptiPrep® in a 5 ml centrifuge tube (TLS 55 Beckman rotor for table top Beckman ultra centrifuge). To avoid mixing the different percentages of OptiPrep® solutions when pipetting the gradient, the solutions are best pipetted by letting them run down the tube wall while holding the pipette tip just above the meniscus of the solution in the tube. A clear line between each percentage of OptiPrep® solution should be visible when holding a black paper behind the centrifugation tube. These clearly separated ‘steps’ of the gradient solutions separate the proteins into sharp bands after centrifugation (instead of a broader fuzzy band). A protocol detailing how to establish a continuous gradient is provided by the vendor.
The centrifugation times for OptiPrep® gradients recommended by the vendor are shorter than those published for most sucrose gradients. For my application, I centrifuged for 2 hours which was sufficient to obtain a good separation of lipid rafts into a band visible in the interphase between the 5% and 35% OptiPrep® solutions. Collection of the fractions can be achieved by slowly pipetting the solution from the top of the gradient or by pinching a whole in the bottom of the tube and collecting the desired fraction volume.
For downstream applications (e.g. immunoprecipitations) with the different fractions, dilution of the fraction is recommended. This is because diffusion of antibodies and larger protein complexes is limited in Optiprep which is has higher viscosity than buffers used in immunoprecipitations and other downstream applications. The proteins in the OptiPrep® fractions can be directly analyzed in SDS-PAGE. Using this method, I obtained reliable and consistent separation of proteins into the same fractions.
Excellent and useful information about OptiPrep® applications can be found on the vendors website. An application brochure as well as the packing insert are available as PDF files. The application brochure describes in detail the properties of OptiPrep®, gradient protocols and isolation methods. Additional technical protocols and extensive references (PDF files) organized by application also provide useful information about different protocols.
OptiPrep® is an easy-to-use gradient media that allows reliable and consistent protein separation.