The best yield (2-3 mg/200 ml) was obtained after the purification procedure described in Fig. 1a, panel B. In comparison, the method described in Fig. 1a , panel C, yielded a ten times lower amount of NoV capsid VLPs. The purity of the VLPs obtained by each method was determined by 12% SDS-PAGE and staining of the proteins with Page-Blue (Fig. 1b) . 1 lg of the total protein was loaded into each lane. Each method resulted in equally pure protein bands corresponding to the size of the NoV capsid. Residual PEG present in the VLPs, concentrated by PEG dialysis, may have interfered with the protein concentration determination, and Fig. 1 Purification, concentration and characterization of NoV VLPs. a Several purification procedures (A-D) were used, and the purity, morphology, antigenicity and yield of the VLPs were compared. b NoV capsid protein analysis by 12% SDS-PAGE. c Western blot analysis using a human convalescent serum against norovirus GII-4. d EM of purified NoV VLPs observed at a magnification of 930,000. Bar 100 nm. Samples from purification procedures A, B, C, and D correspond to lanes and panels A, B, C, and D, respectively. M protein weight marker this could explain the lower capsid protein concentration observed (Fig. 1b, lane A) . NoV capsid identity and antigenicity of the VLPs were assayed by western blot using human NoV-specific convalescent sera (Fig. 1c) . The results show pure NoV capsid proteins without degradation and with similar antigenicity for each of the purification methods. Next, we determined the morphological integrity and homogeneity of the VLPs by EM (Fig. 1d) . The VLPs obtained by purification with sucrose density gradients followed by dialysis and either ultrafiltration (Fig. 1d, panel B) or ultracentrifugation (Fig. 1d, panel C) were approximately 38 nm in size, with the classical appearance of NoV capsid VLPs. By contrast, CsCl purification (Fig. 1d, panel D) and concentration of the VLPs by dialysis against PEG (Fig. 1d , panel A) resulted in VLPs of heterogeneous size, which appeared broken and aggregated. The VLPs purified and concentrated by the method schematically presented in Fig. 1a, panel B , were of the best quality and were subjected to further analysis.