We investigated whether it was possible to measure fumaric acid in faeces. We tried several concentrations of faeces, and the results showed that it is possible to measure fumaric acid in faeces, although the measurement needs to be optimized.
Here is our protocol:
A Fumarate Detection Kit (Abcam 102516) was used for fumaric acid measurement in faeces. 40 mg of faeces was weighed and 100 ul Assay Buffer was added to the samples. Bead-beating (2 times 30 seconds) was performed, and afterwards centrifugation 10 minutes (13.000pm) for homogenization of the samples. The protein concentration of the supernatant was measured with the DC kit from Bradford Assay. Optimum protein concentration was determined testing various ug protein concentrations. After a couple try-outs, it appeared that 2.5 ul and 0.5 ul of the supernatant were the best concentrations to use in the assay. 2.5 ul and 0.5ul were both tested, and added to every well. Assay buffer was added to end up with a total concentration of 50 ul. Consequently, 100 ul reaction mix was added to each sample, which consisted of 90 ul Fumarate Assay Buffer, 8 ul Fumarate Developer and 2 ul Fumarate enzyme mix. After incubation of 60 minutes at 37°C, the absorbance was measured at 450 nm in a microplate reader.
Here is our protocol:
A Fumarate Detection Kit (Abcam 102516) was used for fumaric acid measurement in faeces. 40 mg of faeces was weighed and 100 ul Assay Buffer was added to the samples. Bead-beating (2 times 30 seconds) was performed, and afterwards centrifugation 10 minutes (13.000pm) for homogenization of the samples. The protein concentration of the supernatant was measured with the DC kit from Bradford Assay. Optimum protein concentration was determined testing various ug protein concentrations. After a couple try-outs, it appeared that 2.5 ul and 0.5 ul of the supernatant were the best concentrations to use in the assay. 2.5 ul and 0.5ul were both tested, and added to every well. Assay buffer was added to end up with a total concentration of 50 ul. Consequently, 100 ul reaction mix was added to each sample, which consisted of 90 ul Fumarate Assay Buffer, 8 ul Fumarate Developer and 2 ul Fumarate enzyme mix. After incubation of 60 minutes at 37°C, the absorbance was measured at 450 nm in a microplate reader.
The reviewer received a reward from Abcam’s Loyalty Program in thanks for submitting this Abreview and for helping the scientific community make better-informed decisions.
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投稿 Jun 17 2016