Proteins analysis and nitrogen content measurement
Proteins were separated by sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS–PAGE) on a 15% acrylamide separating gel and a 3% acrylamide stacking gel at 25 mA for 1 h (19), and 4 µg of protein sample was loaded onto each lane of the SDS–PAGE gel (17) and after electrophoresis the gel was subsequently stained with a solution of Coomassie Blue R-250 for 20 min and de-stained for several times with the mixture solution of 125 ml of methanol, 37.5 ml of acetic acid and 337.5 of distilled water.
A nitrogen analyzer (LECO FP-258) was used for nitrogen analysis based on the combustion by oxygen gas. The rubber sample (0.25 g) was weighed and subjected to the nitrogen analysis. The combustion of rubber sample converts the nitrogen in compounds to nitrogen gas, which can then be detected as nitrogen content (% w/w). In this experiment, EDTA was used as a standard with an accuracy of ± 0.02% (w/w). The results were obtained from triplicate analysis.