I dilute sample two time first: when extract by (1) 25 mM Phosphate buffer and second: in(2) 50 mM Phosphate buffer contained 0.25 mM ascorbic acid and 10 mM H2O2 .
if I will dilute third time in 1 or 2 buffer up
I used H2O2 concentration 3%
and what polarographic method did you have assay
sent to me please
We prefer potassium phosphate buffer. Molarity of extraction buffer must be lower than reaction buffer. We use 50 mM for extraction and 100 to 200 mM for reaction buffer (depending on the proportion of crude enzyme extact one prefers to use for final reaction i.e. for determining enzyme activity)
we used 25 mM potassium phosphate buffer (pH 7.8) for extraction and 50 mM potassium phosphate buffer for reaction, but rsult of spectrophotometer was not stabile for more than 3 minutes why?
Your buffers are fine. The basic reason for instability is due to oxygen bubbles that are stuck on the sides of cuvette and interfere with transmission of light. It looks that you have good catalase activity in your samples. Why do you want to go beyond 3 minutes for measurements? You can restrict your measurements to just one or two minutes. If your samples show high catalase activity, please dilute samples.
I did the test ,but instabil in spectrophotometer Once again ?
How I removal oxygen bubbles and How I dilute samples
Please
Please dilute enzyme extract with buffer (may be ten times). How many times did you dilute? Please use lesser concentration of H2O2 (try to use 5 - 10 times lowe concentration of waht you are using now).
Because of this problem, we often prefer using polarographic method. If you have a facility, try to use this method (it is a very simple protocol).
I dilute sample two time first: when extract by (1) 25 mM Phosphate buffer and second: in(2) 50 mM Phosphate buffer contained 0.25 mM ascorbic acid and 10 mM H2O2 .
if I will dilute third time in 1 or 2 buffer up
I used H2O2 concentration 3%
and what polarographic method did you have assay
sent to me please
Please dilute ten times. Do not use ascorbic acid (as it might simultaneously promote ascorbate peroxidase activity).
I am attaching Prof. Walker's book, which gives detailed information on polarographic method. I am also attaching one of our publications where we used this method.
By using potassium phosphate buffer at 50 mM.
Please visit the paper of Vanacker et al. (2000). Plant Physiol. 123: 1289-1300.
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