Fresh mitochondria were isolated from mouse lung homogenate via differential centrifugation in isolation buffer as described earlier [20 (link)]. Mitochondrial preparations (n = 5 per treatment) were incubated with control (0 μM Cd) or CdCl2 (0.001, 0.01 and 1 μM) in incubation buffer (220 mM mannitol, 2 mM HEPES, pH 7.0) for 20 min at room temperature. Excess Cd was then removed by centrifugation at 10,000 g for 1 min. Pelleted mitochondria were resuspended in assay buffer provided by the enzyme activity kit (Sigma-Aldrich, St. Louis, MO). Activity of malate dehydrogenase and isocitrate dehydrogenase was measured following manufacturer’s instructions and normalized to total protein content.
Enzyme activity kit
Manufactured by Merck Group
The Enzyme Activity Kit is a laboratory instrument designed to measure the activity of enzymes. It provides a standardized method for quantifying the catalytic function of enzymes, which are essential biomolecules that facilitate chemical reactions in living organisms.
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Lab products found in correlation
2 protocols using enzyme activity kit
1
Mitochondrial Enzyme Activity Assay
2
Quantification of Thioredoxin Reductase Activity
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For the measurement of thioredoxin reductase (TrxR) activity in HepG2 and MIA PaCa-2 cells we used an enzyme activity kit from Sigma-Aldrich (product#: CS0170). Frozen samples collected during our dose-response experiment (paragraph 2.9) were used to also determine TrxR activity. The samples chosen were: no SLM added to the media to determine basal activity; and 180 nM SLM added to the media to determine a potential increase in activity. The kit indicates that users should normalize the activity to a given volume. Thus, the amount of protein each sample is to be equal. For this experiment, we added on the order of 100 μg of cell protein (exact amount was used in calculations) to the assay buffer (section 2.4.3 ) to yield a total volume of 50 μL. These were then used to measure the activity as described in the protocol of the kit.
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