Precipitation of specific proteins by freeze-thawing of human saliva

Frozen saliva samples demonstrate a variable amount of precipitate on thawi ng depending on the type of secretion [submandibular-sublingual (SML) great er than parotid]. This precipitate has been resuspended using EDTA or remov ed by centrifugation by some workers and others do not mention it. Yet othe rs collect the salivas into EDTA or centrifuge them before freezing. To det ermine the adsorption of proteins to hydroxyapatite, prior treatment with E DTA would be disadvantageous. The aim here was to determine if the protein pattern in parotid and SML saliva as demonstrated by sodium dodecyl sulphat e gel electrophoresis is affected by the formation of precipitates. Portion s of parotid and SML saliva were thawed and treated in the following ways: (a) mixed vigorously with a vortex mixer; (b) centrifuged to remove the pre cipitate: (c) mixed with EDTA (1 and 5 mmol final concentration for parotid and SML samples, respectively) to resuspend the precipitate. The samples w ere loaded on to gradient (5-20%) SDS gels and, following electrophoresis, the gels were stained with Coomassie brilliant blue R-250. The protein patt erns obtained for (a) and (c) were the same. The centrifuged samples demons trated loss of a specific band of less than 14 kDa, although this was less obvious in the parotid samples. The SML samples also showed a reduction in other lower molecular-weight proteins. This study demonstrates that precipi tates in thawed frozen salivas contain specific proteins and that these sam ples require careful handling to avoid any alteration in the overall protei n composition. (C) 2000 Elsevier Science Ltd. All rights reserved.