to evaluate SOD in tissue , we use potassium phosphate buffer with EDTA to homogenize the tissue. i need the amount of both pot.and EDTA solutions /ml
prepare potassium phosphate buffer and EDTA like this
216 mM Potassium Phosphate Buffer, pH 7.8 at 25°C (Buffer)
10.7 mM Ethylenediaminetetraacetic Acid Solution (EDTA)
Usually 50 mM pot. phosphate buffer is used for most of the biological assays. The pH of the buffer depends upon which organism you use for your study. For mammalian system the pH is 7.4. In marine invertebrates the pH is usually higher than 7.4. So you have to accordingly choose the pH. Check the pH of the homogenate after homogenizing in 50 mM pot. buffer. If the pH is fluctuating than your pH of the buffer then increase the strength of your buffer i.e. increase the concentration of your buffer may be to 75 mM or 100 mM. And repeat the above process. The homogenate pH should match the pH of your buffer.
To optimize the assay mixture pH for SOD activity measurement, make a homogenization (may be 10%) of your tissue in deionised distilled water and measure the pH of the homogenate. If you obtain the pH value as "x" then measure SOD activity at pH x+0.5, x+1.0, x+1.5, x-0.5, x-1.0 and x-1.5. You may get the highest SOD activity at any of the above pH. Stick to that pH to be used for your SOD activity measurement. You have to also take care of the temperature. Usually all the above process other than homogenization and centrifugation, is done at 25°C.
EDTA is usually used for divalent metal chelation. In the assay mixture of SOD, EDTA is also used if the assay system uses the principle of color production by riboflavin or nitro blue tetrazolium. Usually 1 mM EDTA is used for both the above processes.
You can search the below link for further information
http://www.sciencedirect.com/science/article/pii/S1532045609002105
iI was means that the amount of solutions of pot.and EDTA we can mix to have the buffer. Now, I knew that I can prepare pot. Buffer with the needed molarity and added EDTA powder to it.
No, EDTA does not hold any part of pot. phosphate buffer. Pot. phosphate buffer means the solution containing K2HPO4 and KH2PO4 in a specific ratio. EDTA is usually added in the buffer before you adjust the pH and volume of your buffer.
As there are many EDTAs, so which EDTA we need to use, Disodium EDTA or simple EDTA?
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