Protein biomarkers of external root resorption: a new protein extraction protocol. Are we going in the right direction?

The aim of this study is to determine a protocol of gingival crevicular fluid protein extraction used for the first dimension of 2-DE gels. It also aims at conducting a review on the current candidates for protein markers of this pathology, all of which may be used to prevent the disease. Gingival crevicular fluid was collected from two groups of 60 patients each, with and without external root resorption. Samples were extracted by means of various methods of protein extraction. SDS-PAGE gels were used to assess the quality of the method which was subsequently tested during isoelectric focusing of 2-DE gels taken from samples of patients with and without the disease. Milli-Q ultrapure ice cold water, without precipitation for gingival crevicular fluid protein extraction, proved the method with greatest sharpness to detect protein bands. Additionally, it allowed two-dimensional electrophoresis to be performed. The new protein extraction protocol does not interfere in isoeletric focusing of 2-DE gels. Furthermore, it provides the greatest sharpness in detecting protein bands of SDS-PAGE gels. This will allow mapping and searching of new external root resorption markers, particularly due to the difficulty in carrying out molecular tests with the current candidates for protein markers.