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1. Start with two 15 cm dishes of cells, grow to 75-80% confluence.
6. Heat both tubes at 95°C for 10 min.
7. Chill on ice then centrifuge for 10 sec.
8. Add the following to each tube:
The total volume in each tube is 660 μL.
9. Incubate both tubes at 37°C for 2 hr.
10. Stop the reaction by adding 330 μL 3X SDS+DTT loading buffer (Blue Loading Buffer Pack #7722). The total volume in each tube is 990 μL.
11. Heat at 95°C for 5 min prior to loading.
What is your protocol for PNGase F treatment?
PNGase F cell treatment:
To deglycosylate a protein of interest in native conditions.
Reagents:
PNGase F, NEB P0704L (https://www.neb.com/en-us/products/p0704-pngase-f)
To deglycosylate a protein of interest in native conditions.
Reagents:
PNGase F, NEB P0704L (https://www.neb.com/en-us/products/p0704-pngase-f)
1. Start with two 15 cm dishes of cells, grow to 75-80% confluence.
2. Lyse each with 250 μL 1X cell lysis buffer (Cell Lysis Buffer (10X) #9803).
3. Combine these two dishes of cells into one tube.
4. Sonicate the lysate.
5. Set up two 1.5 mL tubes:
| Treated Cell Lysate: | Control Cell Lysate: |
| 240 μL cells in lysis buffer | 240 μL cells in lysis buffer |
| 30 μL 10X denaturing buffer | 30 μL 10X denaturing buffer |
| 30 μL water | 30 μL water |
6. Heat both tubes at 95°C for 10 min.
7. Chill on ice then centrifuge for 10 sec.
8. Add the following to each tube:
| Treated Cell Lysate: | Control Cell Lysate: |
| 60 μL 10X GlycoBuffer2 | 60 μL 10X GlycoBuffer2 |
| 60 μL 10% NP40 | 60 μL 10% NP40 |
| 180 μL water | 240 μL water |
| 60 μL PNGase F enzyme |
The total volume in each tube is 660 μL.
9. Incubate both tubes at 37°C for 2 hr.
10. Stop the reaction by adding 330 μL 3X SDS+DTT loading buffer (Blue Loading Buffer Pack #7722). The total volume in each tube is 990 μL.
11. Heat at 95°C for 5 min prior to loading.
Last updated: September 12, 2024
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