Apr 24, 2026
Endo H/ PNGase F Assay for JESS Automated Western Blot
- Charis Ma1,
- Yu Chen1,
- Ellen Sidransky1
- 1National Human Genome Research Institute
Protocol Citation: Charis Ma, Yu Chen, Ellen Sidransky 2026. Endo H/ PNGase F Assay for JESS Automated Western Blot . protocols.io https://dx.doi.org/10.17504/protocols.io.5jyl88q18l2w/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 17, 2025
Last Modified: April 24, 2026
Protocol Integer ID: 230104
Keywords: jess automated western blot system, jess automated western blot, western blot, assay, endo, protocol detail
Funders Acknowledgements:
ASAP
Grant ID: ASAP-024297
Abstract
This protocol details an optimized Endo H and PNGase F digestion assay that produces samples that can be run with the JESS Automated Western Blot System.
Materials
10X Glycoprotein Denaturing Buffer (NEB, Catalog# B0701S)
10X GlycoBuffer 3 (NEB, Catalog# B0701S)
10X GlycoBuffer 2 (NEB, Catalog# B0701S)
10% NP-40 (NEB, Catalog# B0701S)
EndoH (NEB, Catalog# P0703S)
PNGase F (NEB, Catalog# P0704S)
10K Amicon Ultra Centrifugal Filter (Millipore, UFC5010BK)
Glycoprotein Denaturation
In an eppendorf tube, add 40 µg protein, 2 µL 10X Glycoprotein Denaturing Buffer, and water for a total reaction volume of 20 µL . Mix well.
Denature glycoprotein by heating reaction at 100 °C for 00:10:00 .
10m
Split reaction volume into 2 eppendorf tubes with 10 uL in each tube. One tube will be used for the EndoH reaction, while the other will be used for the PNGaseF reaction.
Endo H
Add the following to one reaction tube from step 3: 2 µL 10X GlycoBuffer 3, 2 µL Endo H, 6 µL water. Total volume will be 20 µL .
Mix gently.
Incubate reaction at 37 °C for 01:00:00
1h
PNGase F
Add the following to the second reaction tube from step 3: 2 µL 10X GlycoBuffer 2, 2 µL NP-40, 6 µL water, 1 µL PNGaseF. Total volume will be 21 µL .
Mix gently.
Incubate reaction at 37 °C for 01:00:00 .
1h
Desalting and Washing for JESS Automated Western Blot System (Bio-Techne) preparation
Add 480 µL and 479 µL of 0.1X Sample Buffer (Bio-Techne) to the Endo H and PNGaseF reaction tubes respectively to make the final volume 500 µL .
Add all 500 µL of each reaction to a 10K Amicon Ultra Centrifugal Filter
Spin at 14000 x g for 00:20:00 . You will recover a final volume of20 µL , and the protein concentration recovered will be ~1 mg/mL .
20m
JESS Sample Preparation
5m
Add 4 parts of the washed Endo H/ PNGaseF sample from step 12 with 1 part of 5X Fluorescent Master Mix (Bio-Techne).
Vortex to mix
Incubate sample at 95 °C for 00:05:00 .
5m
Load into JESS microplate (Bio-techne) to be detected by the Automated Western Blot System.