Modified PNGase Protocol

(Denaturing Reaction Conditions:)

1. Combine cell pellet (approximated to be several mg) with 100 µL of the

Glycoprotein Denaturing Buffer mix.

Original protocol says to mix 1 µL of Glycoprotein Denaturing Buffer (10x) and water to
make a total reaction volume of 10 µL with 1-20 µg of glycoprotein. Marcel advises
estimating the amount of glycoprotein based on the height of the cell pellet.
1 mm = 1 mg.

Estimates:
C5 5 mg
C8 3 mg
D10 2 mg
F2 4 mg
G8 5 mg
G9 1 mg

Clones C5, C8, D10, and G9 were selected to analyze preliminarily. To ensure that
enough reaction mix was added to these pellets, Marcel advised simply adding 100 µL
of the reaction mix to each pellet.

So, a master mix of 50 µL Glycoprotein Denaturing Buffer and 450 µL of water was
prepared. 100 µL of this mix was added to each cell pellet

2. Using a blue Eppendorf “stick”, lyse the cell pellet by spinning the stick against
the bottom of the Eppendorf tube for 10 min. The resulting pellet should become
viscous/sticky/’glue-y’.
3. Denature the lysates by heating the tubes at 100 deg Celsius for 10 min. The 95
deg Celsius heat-block was used per Marcel’s recommendation.
4. Chill on ice for 10 min.
5. Centrifuge the tubes at 5,000 RPM for 5 min.
6. Transfer the supernatant (containing the surface glycoproteins) to a new
Eppendorf tube, being careful not to perturb the pellet of cellular debris.
7. Store on ice. Proceed with the Bradford Assay to quantify the amount of protein
in the lysates.