Western blot analysis of the expression of b 2 microglobulin, a enolase, immunoglobulin k light chain and a amylase fragmentation pattern The WB was used to ensure the reliability of the 2DE results. In particular, we investigated the expression of download catalog b 2 microgloblulin, a enolase, immunoglobulin k light chain and the a amylases. In order to character ise the fragmentation pattern of a amylases, 2D blots were performed using specific antibody direct versus the full length of human recombinant protein in all the groups. In the process of 1D WB, aliquots of samples were mixed with a SDS sample buffer and heated at 100 C for five minutes. The WB was carried out as exactly pre viously described. Briefly, aliquots of proteins, extracts from mix pooled WS samples of each class, were loaded on 12% acrylamide gels and processed.

For the protein detection the following antibodies were used, mouse monoclonal anti full length b 2 microglo blulin, mouse monoclonal anti a enolase, duck polyclonal Inhibitors,Modulators,Libraries anti full length a amylases and rabbit polyclonal anti IGKC All the experiments were carried out in tri plicate. For each tested protein, the optical density of specific immunoreactive bands was determined, and the resulting mean values SD were compared. For the detection of a amylases, we chose a specific antibody direct versus a full length of human recombinant albumin, to detect the potential protein fragmentation. Aliquots of 100 ��g of proteins were separated by 2DE using 3 to 10 linear strips 13 cm before Western blot ana lysis. The dilution was 1,500 and 1,10,000 for anti a amy lases primary antibody and anti Inhibitors,Modulators,Libraries Inhibitors,Modulators,Libraries duck, respectively.

To Inhibitors,Modulators,Libraries assure a correct control of development conditions a solution of standard proteins was loaded in each experiment and the time and temperature of the process were controlled. ELISA ELISA were used to determine the level both of b 2 microglobulin and a enolase in saliva sam ples from healthy, pSS, non SS sicca syndrome and sSS subjects. Whole sal iva samples were thawed, vortexed and centrifuged to eventually remove mucins precipitation prior to assay ing. Saliva samples were diluted with 20 mM PBS buffer, pH 7. 2 at a final dilution of 1,100 and 1,5 for b 2 micro globulin and a enolase, respectively. The assay was per formed according to the manufacturers instruction manual.

Samples were analysed in duplicate, Inhibitors,Modulators,Libraries and the protein levels were determined according to the calibra tion curves established from standards. Salivary a amylase assay For a amylase a salivary a amylase assay kit specifically designed and validated for the kinetic measurement of salivary a amylase activity was used. Saliva sam ples were diluted with a amylase diluent provided at a final dilution of 1,200. Aliquots of 8 ul of diluted sam ples were added to individual wells and the reaction was started by www.selleckchem.com/products/Perifosine.html the simultaneous addition of preheated a amylase substrate solution.