lost of enzyme activity after thawing - (May/28/2007 )
Now, I'm been working with EGFR (tyrosine kinase) in ELISA-based assay (the kit from Chemicon)
The fisrt time I tested, the activity of enzyme was very good. But the from the second time, I saw almost no activity of enzyme. I has tested again several times and the results were the same (very week activity).
I wonder just one thaw-freeze cycle can destroy almost the activity of enzyme or any other reason but I dont recognize?
If anyone have some experience in this field, please give me some advice: how to thaw and freeze to reduce the effect on activity of enzyme and also other technique should be noticed.
Thanks in advance!
The fisrt time I tested, the activity of enzyme was very good. But the from the second time, I saw almost no activity of enzyme. I has tested again several times and the results were the same (very week activity).
I wonder just one thaw-freeze cycle can destroy almost the activity of enzyme or any other reason but I dont recognize?
If anyone have some experience in this field, please give me some advice: how to thaw and freeze to reduce the effect on activity of enzyme and also other technique should be noticed.
Thanks in advance!
I miss some more details in your description; what is your biological material and how does your ELISA work (phospho EGFR detection?)
I think you work with enriched plasma membranes or yesicles;
freeze-thawing cycles do harm to most enzymes; in your case, there is also another problem: freeze-thawing changes the ratio of inside-in to inside-out vesicles
This time, I tested with commercial purify enzyme.
And we used ELISA to dectect phosphopetide (that created in kinase reaction)
And we used ELISA to dectect phosphopetide (that created in kinase reaction)
isolated membrane proteins especially those with enzyme (here: kinase) or channel function are very critical towards stability if not reconstituted in their membrane lipid phase