For determination of ROS in mitochondrial fraction using DCFDA, How to quantify the concentration of isolated mitochondrial fraction? using nanospectrometer or some other technique ?
If i perform BCA then it will give protein concentration in mitochondria....
But in that protein fraction, i don't think ROS production would b getting accessed.
So, if i hv isolated mitochondrial fraction, then how can i quantify its concentration?
As for DCFDA, i need some specific concentration of mitochondrial fraction (per se: 1mg/ml), so how to determine that concentration?
as in protocol they hv not mentioned anywhere that i need to hv mitochondrial protein fraction (1mg/ml)...they just said: mitochondrial fraction...
so without BCA how can i quantify my isolated mitochondrial fraction easily and quickly so that i can proceed my mitochondrial fraction for further experiments like ROS or membrane potential etc...
kindly confirm
Hello Asha,
Let me clear you that the DCFH-DA works in the presence of intracellular esterase, hence the intact live cells are used to analyze intracellular H2O2 and other ROS. Indeed, DCFH-DA is commonly used for detecting intracellular H2O2.
DCFH-DA is a cell-permeable ester and is hydrolyzed inside the cell to the dihydroxy-DCFH, which is retained. Despite the popularity of this assay, it cannot be reliably used to measure intracellular H2O2 and other ROS for the following reasons: (i) DCFH does not directly react with H2O2 (ii) several one-electron oxidizing species will oxidize DCFH to DCF (iii) DCF can actually produce O2•− and H2O2 via reaction of DCF radical with the oxygen, thus artificially elevating the very ROS that it is attempting to quantify; (iv) transition metals, cytochrome c, and heme peroxidases can catalyze DCFH oxidation. Hence, due to these reasons, DCHF-DA should not be used as a reliable measure of H2O2.
The isolation of mitochondrial fraction is not easy and may have contamination with ER etc. You can have semi-pure mitochondria fraction but it is possible once you rupture cells of your interest and collect the semipure fraction using gradient centrifugation. And ROS is highly reactive, hence it is recommended that measurement (quantification) must be done in cell lysates as early as possible in order to avoid any changes in ROS.
The new method has been developed by Kalyanaraman and colleagues in 2003. Following this original report, DHE has been modified to allow detection of O2•− in the mitochondria by addition of a triphenylphosphonium group, which promotes its accumulation in the mitochondria. This modified DHE analog is referred to as mitoSOX, and has become commonly used for detection of O2•− within the mitochondria. The mitochondrial H2O2 can also be detected by fluorescent protein-based redox probes. There are several other methids by which the mitochondrial ROS level can be analyzed.
To quantify the mitochondrial protein content, normally BCA assay is used but I wonder if the mitochondrial protein content level give you any clue for ROS level and that too in isolated mitochondria fraction. Hence, I would suggest you to analyze the total ROS level using DCHF-DA dye in live cells under fluorescence microscope.
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