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. Author manuscript; available in PMC: 2014 Oct 3.
Published in final edited form as: Curr Protoc Microbiol. 2013 Oct 2;30:Unit–18.2.. doi: 10.1002/9780471729259.mc1802s30

Figure 1. Diagram of sucrose gradients used to purify VLPs.

Figure 1

Large-scale purification of VLPs requires two to three sequential sucrose gradients, which are diagramed. In step one, VLPs, pelleted from the cleared cell supernatants, are resuspended in TNE buffer and placed on top of layers of 65% and 20% sucrose, and then subjected to centrifugation for 6 hours at 24,000 rpm (step 1). The white fluffy layer between the 65% and 20% layers is harvested. In step 2, the VLPs from step 1 are subjected to flotation into a gradient. The VLPs, made approximately 60% with respect to sucrose, are placed on top of an 80% sucrose pad and then over layered with 50% sucrose and then 10% sucrose. The gradients are centrifuged at 38,000 rpm for at least 18 hours. The VLPs form a white layer in between the 50% and 10% layers (step 2). For additional purification, the VLPs can be subjected to sedimentation in a continuous sucrose gradient (Step 3).

Small-scale purification of VLPs can be accomplished by centrifugation of VLPs through a 20% sucrose layer into a pellet as diagramed at the bottom of the figure. Alternatively, step 1 of the large-scale purification can be done.