Quantification of MMP-8 in Mouth Rinse Samples

The molecular forms of MMP-8 were detected from mouth rinse samples by a modified enhanced chemiluminescence (ECL) Western blotting kit according to protocols recommended by the manufacturer (GE Healthcare, Amersham, UK) as described earlier by Rautava et al. [41 (link)]. Briefly, the proteins of mouth rinse samples were first separated by electrophoresis and then electro-transferred onto nitrocellulose membranes Protran (Whatman GmbH, Dassel, Germany). The membranes were incubated overnight with monoclonal primary antibodies anti-MMP-8 [42 (link)] and then with horseradish peroxidase-linked secondary antibody (GE Healthcare, Buckinghamshire, UK) for 1 h. The membranes were washed 4 times in TBST between each step for 15 min. The proteins were visualized using the ECL system according to protocol. The recombinant human MMP-8 (100 ng, Calbiochem, Darnstadt, Germany) was used as a positive control.