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protein deamidation.. glutamine specific protocols?
Posted: Wed Apr 02, 2008 1:24 pm
by alicee!
hi does any one have.. protein deamidation.. glutamine specific protocols? basic and acidic conditions lead to deamidation.. preferably for asn.. but my protein only has gln.. any ideas?
Posted: Thu Apr 03, 2008 8:37 am
by danko
Hi Alicee,
I have deamidated glutamine in different proteins successfully I many occasions.
Just dissolve the protein in 0.02 – 0.04 M HCl and incubate the solution at 50 degrees centigrade for a day or 2.
You’ll need to follow the deamidation analytically in order to find the suitable time and degree of deamidation.
Best Regards
Posted: Thu Apr 03, 2008 1:34 pm
by alicee!
hey thanks danko..
i have been trying the condition u described.. but my protein aggregates as well as fragments in the condition. it may be deamidating but i'm not too confident. how do u confidently say deamidation?
i've been seeing a separate peak in rp-hplc.. but that is obviously not all i can do. how do u do a confirmation? i'm planning on iec.. what else? thanks!!
Posted: Thu Apr 03, 2008 2:37 pm
by danko
Hi again,
I wouldn’t expect any fragmentation under these, relatively mild conditions.
As for the aggregation; it is – beside other factors – concentration dependant (the higher the concentration the higher risk of aggregation). Obviously I don’t know the concentration you’re working with, but if you dissolve your protein to 0.2 – 0.3 mg/ml it should be “saveâ€
Posted: Thu Apr 03, 2008 2:41 pm
by danko
....employ ICE....
Sorry! Make that IEC.
Best Regards