Native PAGE separates the various forms of polymerized C1-inh according to their electrophoretic mobility, in contrast to SDS-PAGE, which, as demonstrated in the present study, dissolves polymeric C1-inh into monomers. Therefore, native PAGE is an attractive method to analyze the size distribution of polymerized C1-inh. A limitation to this method is that MAb was produced against artificially formed C1-inh polymers. This might give rise to false negative results, as in vivo occurring polymers potentially display different epitopes than those ASP1517 808118-40-3 observed in C1-inh polymers formed using heat denaturation. Polymerized C1-inh was prepared by heat denaturation, and was used as marker throughout the experiments. To assure that the molecular weight of the polymer bands observed in native PAGE corresponded to that expected for C1-inh polymers in SDS-PAGE, we conjugated the pC1-inh using the zero-lengthlinker BS3. Using this method, we determined the molecular weight of the BS3-conjugated polymers in SDS-PAGE, and subsequently we compared BS3-conjugated polymers with pC1inh in samples on native PAGE. By this approach we confirmed that the molecular weight of pC1-inh, as observed in native PAGE, corresponded to the molecular weight of BS3 conjugated polymers. Furthermore, we prepared a low molecular weight form of the pC1-inh, resembling the polymer size observed in patient plasma samples. PC1-inh, LMW pC1-inh and monomeric C1-inh were used as markers in all native PAGE WB, where patient plasma samples were analyzed. Using this method we detected polymers in patient plasma samples and evaluated the size distribution of the polymeric species. Mutation number 12 was associated with a low molecular weight C1-inh band without a polymerization phenotype. This band probably represents a secreted mutated form of C1-inh, which apparently is not recognized by the anti C1-inh antibodies in the nephelometric methods used for the type I and II classification, as Vismodegib moa patients carrying this mutation are identified as type I patients. It is important to emphasize that the present native PAGE WB method might suffer from similar problems with regard to recognition of mutated C1-inh. We cannot exclude the possibility that the assay does not recognize all C1-inh polymer forms, as the epitope recognized by our MAb might be masked in specific C1-inh mutations. The positions in the peptide sequence of the polymerogenic SERPING1 mutations have been located in the model. Mutation number 6 is located in strand 3A on betasheet A, in the region termed the shutter domain. It is well established that mutations affecting the shutter domain of a1antitrypsin lead to polymerization of this serpin. The underlying mechanisms are suggested to involve opening of the beta-sheet A in the mutated serpin, and subsequently the RCL of another a1-antitrypsin molecule can insert into this domain with ensuing polymer formation. Mutation number 13 results in a deletion in the strand connecting helix F with strand 3 in beta-sheet A. Helix F is positioned in front of the shutter domain, and in this way hinders opening of beta-sheet A, which protects the serpins against polymer formation. We speculate that deletions in the strand connecting helix F and strand 3A of C1-inh result in dislocation of the helix F and opening of beta-sheet A, with subsequent insertion of the RCL from another C1-inh molecule. Mutation number 21 introduces an asparagine residue in helix C of C1-inh, and potentially a new N-glycosylation site in helix C. To our knowledge no mutations affecting helix C in serpins have previously been associated with polymerization. An explanation for this observation is that the mutation may facilitate interactions between helix C and beta-sheet A.