Sep 04, 2024

Public workspaceEndo H/ PNGase F Assay for JESS Automated Western Blot

DOI
https://dx.doi.org/10.17504/protocols.io.4r3l2qzyjl1y/v1
  • Charis Ma1,
  • Yu Chen1,
  • sidranse 1
  • 1National Human Genome Research Institute
Charis Ma
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DOI: https://dx.doi.org/10.17504/protocols.io.4r3l2qzyjl1y/v1
Protocol Citation: Charis Ma, Yu Chen, sidranse 2024. Endo H/ PNGase F Assay for JESS Automated Western Blot . protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2qzyjl1y/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: September 03, 2024
Last Modified: September 04, 2024
Protocol Integer ID: 106891
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)
Troubleshooting
Glycoprotein Denaturation
In an eppendorf tube, add Amount40 µg protein, Amount2 µL 10X Glycoprotein Denaturing Buffer, and water for a total reaction volume of Amount20 µL . Mix well.

Denature glycoprotein by heating reaction at Temperature100 °C for Duration00:10:00 .

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: Amount2 µL 10X GlycoBuffer 3, Amount2 µL Endo H, Amount6 µL water. Total volume will be Amount20 µL .

Mix gently.
Incubate reaction at Temperature37 °C for Duration01:00:00

1h
PNGase F
Add the following to the second reaction tube from step 3: Amount2 µL 10X GlycoBuffer 2, Amount2 µL NP-40, Amount6 µL water, Amount1 µL PNGaseF. Total volume will be Amount21 µL .

Mix gently.
Incubate reaction at Temperature37 °C for Duration01:00:00 .

1h
Desalting and Washing for JESS Automated Western Blot System (Bio-Techne) preparation
Add Amount480 µL and Amount479 µL of 0.1X Sample Buffer (Bio-Techne) to the Endo H and PNGaseF reaction tubes respectively to make the final volume Amount500 µL .

Add all Amount500 µL of each reaction to a 10K Amicon Ultra Centrifugal Filter

Spin at Centrifigation14000 x g for Duration00:20:00 . You will recover a final volume ofAmount20 µL , and the protein concentration recovered will be ~Concentration1 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 Temperature95 °C for Duration00:05:00 .

5m
Load into JESS microplate (Bio-techne) to be detected by the Automated Western Blot System.
Protocol references