Citrate buffer preparation - extraction buffer preparation (Feb/28/2009 )
I am preparing an extraction buffer for peroxidase assay in plant material. According to the method i should extract in 100ml 0.1M (pH 6.2) citrate buffer containing 5% PVPP and 0.4 M calcium chloride.
I managed to prepare 1 liter of 0.1M (pH 6.2) citrate buffer using citric acid and sodium citrate. Then i dissolved the required concentration of calcium chloride and the pH dropped to 4.6. I feel there is an error in my addition of CaCl2. what should i do to maintain the pH at 6.2?
Finally, can i do a POD assay without using PVPP as i see in other methods(currently i don't have it in stock )
"Finally, can i do a POD assay without using PVPP as i see in other methods"
You can always try the assay without PVPP if only get the assay established while you wait for order to arrive (assuming you have plenty of material). However, plant phenolics (if not removed by PVPP or some other method) will likely lower the activity you observe by precipitating your peroxidise and/or directly inhibiting the enzyme. Their antioxidant capacity may also mean you have to increase the amount of H2O2 in the assay.
My best guess for the pH effect is that you have a competitive ion effect, 0.4M seems to be a high amount for an extraction buffer. Hopefully someone will enlighten us .
you should prepare, and adjust the pH of, a concentrated stock of cacl2. use the stock, rather than powder, to make your solution.