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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!

-camellia8881-

QUOTE (camellia8881 @ May 28 2007, 02:54 PM)
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!


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

-The Bearer-

This time, I tested with commercial purify enzyme.
And we used ELISA to dectect phosphopetide (that created in kinase reaction)

-camellia8881-

QUOTE (camellia8881 @ May 28 2007, 04:10 PM)
This time, I tested with commercial purify enzyme.
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

-The Bearer-