hello everyone!!!!!!!!!!!!!!!! I am using PNGase F enzyme(Biolabs) to cleave carbohydrates from the proteins of my interest.Am treating the sample with the enzyme as per manufacturer instructions.
Typical reaction conditions are as follows: 1. Combine 1-20 µg of glycoprotein, 1 µl of 10X Glycoprotein Denaturing Buffer and H2O (if necessary) to make a 10 µl total reaction volume. 2. Denature glycoprotein by heating reaction at 100°C for 10 minutes. 3. Make a total reaction volume of 20 µl by adding 2 µl 10X G7 Reaction Buffer, 2 µl 10% NP40, H2O and 1-2 µl PNGaseF. 4. Incubate reaction at 37°C for 1 hour. But when am doing western blot probed for SAF 32, I could able to see the band at 36 kilo dalton which corresponds to PNGase F. So, what should I do inorder to inactivate or degrade the PNGase F after treating with my samples, so that i could not see the band? OR is there any further step that i should follow before adding the 5X sample buffer to treated sample and going for SDS-PAGE.? Please can anybody help me in this regard. waiting for replies. thank you in advance. divya. _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
