regarding the PNGase F treatment

divya teja via methods%40net.bio.net (by divya.medbio from gmail.com)
Tue Jul 24 11:28:45 EST 2012


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.


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