protein deamidation.. glutamine specific protocols?

[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?

[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

[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!!

[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€

[danko]

....employ ICE....

Sorry! Make that IEC.

Best Regards