Molecular analyses

Molecular assessment will include research regard to detection whether dMmp1 and dMmp2 from brain of adult males of Drosophila fragile X model are involvement in pharmacodynamic pathways of polyphenols.

Western blot analysis

The heads of all flies used in previously described experiments will be removed and placed on dry ice. The heads will be divided in groups in accordance with experimental groups. Chemiluminescent imaging system, and quantitative measurements of Western blots will be performed using the adequate software. Western blots results of control and experimental samples will be compared by statistical software.

SDS-PAGE gelatin and casein zymography

One microgram of purified MMP1 and MMP2 will be used for gelatin and casein zymography, respectively.

For gelatin zymography, protein samples will run on 10% Zymogram Plus (Gelatin) Protein Gels with Zymogram Running Buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3). Electrophoresis will be performed under non-reducing conditions at 24 °C. For casein zymography, a 10% SDS-polyacrylamide gel containing 1 mg/ml casein will be made and run under the same conditions.

After electrophoresis, the gel will be washed twice for 1 h in of 2.5% Triton X-100 (v/v) to remove SDS, then incubated for 24 h at 37 °C in 1 × Zymogram Development Buffer. After incubation, the gel was stained with Coomassie Brilliant Blue to visualize the transparent lytic bands.

MMP inhibition test on zymography

In order to verify that the clear zones resembling matrixmetalloproteinase, polyphenols will be added into the samples before incubation to inhibit MMP1 and MMP2 activities on casein and gelatin zymography. Results of control and experimental samples will be compared by statistical software.

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