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Thank you for the reply. I mean the aconitase kit ab83459. There is a formula in the section of data analysis.
Good thing about this kit is that a little amount of tissue is necessary for analysis, in my case it would be brain tissue and cell culture.
But another thing that confuse me about this kit is that there is an activation buffer (not preservation one) and the compound with iron, becouse as far as I know according to articles this enzyme might be activated. The point for me is to measure aconitase activtity in the samples considering the sensitivity of this enzyme to ROS, thus it means that aconitase is a sensor of oxydative stress and a tool to estimate the oxydative status of the cell.
Actually the second kit ab109712 seems suitable, but the advisable protein concentration is 5 mg/ml, the problem is that I have less protein concentration (when I have experiments with the particular brain region, there is a really small amount of tissue). how much does the protein concentration affect the assay? I know that there should be some limit. And can the homogenate be used?
The good thing in this kit is the preservation solution instead to activation one.
And speaking about this second kit - the calculations is confusing for me, actually there is no formula, just an example
Example: 12.5 μg bovine heart mitochondria
Background: 0.4 mOD/min,
Corrected rate: 38.1±1.3 mOD/min
= 17.3 μM/min
0.25 mL/well = 4.3 nmoles/min
12.5 μg /well = 0.35 μmoles/min/mg mitochondria
Extinction coefficient = 2.2 OD mM-1/well*
So, it means to take 3 OD of the sample from linear part of the kinetic curve, calculate the average rate, then minus background, then divide by sample volume and Extinction coefficient, is it wright?
Thank you in advance,
Asked on Aug 21 2012
Thank you for your interest in our products.
I have been discussing your enquiry with my colleagues in the Lab and would like to share the following information:
Regarding ab109712: Aconitase Enzyme Activity Microplate Assay Kit
I can confirm that a homogenate can be used but it needs to be very concentrated (no less than 2mg/mL). There is no need to extract mitochondria so long as the homogenate is concentrated. You must ensure that the tissue is NOT homogenized in the preservation buffer (which is only required for cells). We have tested in the past Rat liver tissue homogenized in 10mM Tris pH 7.4 + 250mM Sucrose buffer +0.2mM EDTA (you may add 2mM sodium citrate and/or 2.5mM MnSO4 during the homogenization procedure to maintain a stable aconitase enzyme). From personal experiencewe have found that, at least in Rat liver homogenate stored at -80C at a concentration of 25mg/mL and solubilized at 5mg/mL, Citrate and MnSO4 do not improve on the aconitase signal obtained with the simple sucrose buffer. However these additives may be useful in other tissues/homogenates stored at lower concentration.
The protein concentration is important for optimal performance. One main reason is that for optimal signal (at least for rat liver homogenate) samples should be loaded at 100ug/50uL (2mg/mL) and therefore a calibration curve should be set to go above and below is concentration. There is also the issue of protein to detergent ratio during the solubilization of the homogenate.
For the calculations =
experiments should be run in triplicates on kinetic mode reading milliODs and not ODs.
Always run a background (no sample)
Always run a calibration curve. For Rat liver homogenate for example the limit of detection has been found at 100ug/mL (50uL loading prior to the addition of 200uL of activity buffer) and the range has been found between 7mg/mL to 0.1mg/mL (again in the 50uL loading). This would change from tissue to tissue.
Data will come out of the instrument in mOD/min, this can be divided by the extinction coefficient to obtain the molar equivalency.
Regarding ab83459: Aconitase Assay Kit
Yes, this kit comes with an activation solution components instead of preservation ones. Aconitase preservation solution will maintain aconitase activity during sample preparation whereas an inactivated aconitase may be activated in vitro by the addition of iron and cysteine.
Exposure of aconitase to oxidants renders the enzyme inactive and loss of aconitase activity in cells or in biological samples treated with pro-oxidants has been interpreted as a measure of oxidative damage.
It is this aconitase which will be activated for measuring with this kit.
Hope this information has been helpful for you.
Answered on Aug 21 2012