TITLE: Endoglycosidase H (Endo H) digestion assay
AUTHORS: Vik Khurana

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol details the endoglycosidase H (Endo H) digestion assay.</div></div>

[STEPS]
?. [Endoglycosidase H (Endo H) digestion assay]
PREPARE:
?. [Endoglycosidase H (Endo H) digestion assay]
Make sure the lysis buffer (Table 1) is chilled.
?. [Endoglycosidase H (Endo H) digestion assay]
Get samples and keep on dry ice.
?. [Endoglycosidase H (Endo H) digestion assay]
Get a large box and fill to approximately 2/3 with wet ice for the ice/water slurry (see below).
?. [Endoglycosidase H (Endo H) digestion assay]
Prepare lysis buffer MM: Add 100X protease inhibitor cocktail (“PiM”) as well as 100X phosphatase inhibitor cocktail I (“PP”) and II to the lysis buffer in a 1:100 ratio. Keep the lysis buffer MM  from this point onwards. The protease inhibitor cocktail can be briefly thawed using the  heat block. Keep at  before re-freezing.
on ice
37 °C
37 Room temperature
?. [Endoglycosidase H (Endo H) digestion assay]
Cool down ultracentrifuge to . Set parameters: TLA-100 rotor, , , ; apply vacuum.
4 °C
Centrifuge: 100000 34
4 °C
?. [Endoglycosidase H (Endo H) digestion assay]
When ready, thaw samples on wet ice and add an appropriate volume of the lysis buffer MM as soon as the cell pellet is almost thawed.
Can also be thawed briefly at   but make sure that the cell pellet is not completely thawed when lysis buffer MM is added.Lysis buffer MM volume: approximately  - ; around 3x the volume of the cell pellet; better too little than too much.
?. [Endoglycosidase H (Endo H) digestion assay]
Resuspend the cell pellet to a homogenous (!) solution by trituration and keep on wet ice.
Make sure the top of the Eppis is carefully labeled since side labels will likely disappear during a later incubation in the dry ice/EtOH slurry!.
?. [Endoglycosidase H (Endo H) digestion assay]
Prepare the ice/water slurry: add cold water to approximately half of the height of the ice layer.
?. [Endoglycosidase H (Endo H) digestion assay]
Incubate samples for  in the ice/water slurry.
?. [Endoglycosidase H (Endo H) digestion assay]
In the meantime  PREPARE the following:
?. [Endoglycosidase H (Endo H) digestion assay]
Label ultracentrifuge tubes on the side. Use P200 tip holder as a rack for the ultracentrifuge tubes.
?. [Endoglycosidase H (Endo H) digestion assay]
Prepare dry ice/EtOH slurry approximately  before the  incubation on the ice/water slurry is completed:Add dry ice until saturation (dry ice doesn’t resolve quickly anymore; temperature of has stabilized).
-80 °C
?. [Endoglycosidase H (Endo H) digestion assay]
Put Eppis into a floating rack and immediately place them in the dry ice/EtOH slurry in order to snap freeze the samples; incubate for .
Make sure that the lid does not touch the dry ice/EtOH slurry to prevent EtOH leaking into the Eppis; only cell lysate needs to be exposed to the dry ice/EtOH slurry. Incubation time of   is minimum; can incubate longer.
?. [Endoglycosidase H (Endo H) digestion assay]
Directly transfer Eppis onto a heat block and incubate at  for exactly (!)  (no longer!).
37 °C
?. [Endoglycosidase H (Endo H) digestion assay]
Repeat the described freeze/thaw cycle once → two freeze/thaw cycles (/) in total.
-80 °C
37 °C
?. [Endoglycosidase H (Endo H) digestion assay]
Keep samples on wet ice afterwards.
?. [Endoglycosidase H (Endo H) digestion assay]
Get TLA-100 rotor (Beckman Coulter) and keep on wet ice, including the lid. TLA-100 rotor usually kept in the  cold room.
4 °C
?. [Endoglycosidase H (Endo H) digestion assay]
Transfer the cell lysate from the Eppis into the labeled ultracentrifuge tubes (thickwall polycarbonate tubes) and place them in the TLA-100 rotor ; keep the empty Eppis for a later step in case the labeling is sufficient and hasn’t been removed by the EtOH.
on ice
?. [Endoglycosidase H (Endo H) digestion assay]
Place TLA-100 rotor into the ultracentrifuge; make sure it’s correctly placed (!); re-apply vacuum, make sure that the parameters are set correctly ( for  at ) and start. The ultracentrifuge might take a while to start because it will have to re-establish the vacuum; make sure that the ultracentrifuge gets back to .
Centrifuge: 100000 34
4 °C
4 °C
?. [Endoglycosidase H (Endo H) digestion assay]
During the ultracentrifugation  PREPARE the following:
?. [Endoglycosidase H (Endo H) digestion assay]
Get TLA-100 rotor and keep on wet ice.
?. [Endoglycosidase H (Endo H) digestion assay]
Remove ultracentrifuge tubes and transfer the supernatant to a new, correctly labeled and chilled Eppi; keep Eppis on wet ice.
?. [Endoglycosidase H (Endo H) digestion assay]
A small cell pellet should be visible; transfer to the appropriate old, correctly labeled Eppi and keep on dry ice.
This cell pellet can be re-used, e.g., to check for αSyn levels by Western blotting; in this case store cell pellet at   and later resuspend in approximately   of the appropriate lysis buffer for Western blotting.
?. [Endoglycosidase H (Endo H) digestion assay]
For the BCA test either add  of the cell lysate to  water or dilute  of the cell lysate in a 1:1 ratio with  water before adding  of the 1:2 dilution to  water; in the latter case don’t forget to change the dilution factor on the Endo H spreadsheet to 50!.
1 µl
24 µl
2 µl
2 µl
1 µl
24 µl
I got more accurate results with a dilution of 1:25.
?. [Endoglycosidase H (Endo H) digestion assay]
If the Endo H is proceeded with on another day, transfer the Eppis with the cell lysate directly onto dry ice and immediately store them at  (to reuse, thaw cell lysates on wet ice); if the Endo H is to be proceeded with on the same day, place Eppis on wet ice and store intermediately in the  cold room.
-80 °C
4 °C
?. [Endoglycosidase H (Endo H) digestion assay]
During the incubation for the BCA test at  for  roughly label 2 Eppis for each condition (→ +/- Endo H).
37 °C
?. [Endoglycosidase H (Endo H) digestion assay]
Thaw 10X glycoprotein denaturing buffer and 10X G5 buffer at  (don’t vortex denaturing buffer as this will create a lot of small bubbles!).
4 Room temperature
Fine to briefly keep them in the heat block if they are not thawed when needed in the steps below; make sure that these buffers are very well mixed before adding them to the reaction volume!.
?. [Endoglycosidase H (Endo H) digestion assay]
After BCA test use Chee-Yeun’s Endo H spreadsheet to calculate the protein concentration.
?. [Endoglycosidase H (Endo H) digestion assay]
PREPARE: thaw Endo H (stored at ) on wet ice.
-20 °C
?. [Endoglycosidase H (Endo H) digestion assay]
From this point onwards all in duplicate (→ +/- Endo H):
?. [Endoglycosidase H (Endo H) digestion assay]
Thaw sample buffer (“5X SB”, kept at -20°C; Table 2) and protein ladder at .
-20 Room temperature
?. [Endoglycosidase H (Endo H) digestion assay]
In case +/- Endo H samples have been frozen down, thaw at  and briefly spin down.
-20 Room temperature
?. [Endoglycosidase H (Endo H) digestion assay]
Add  5X SB to each  rxn; briefly vortex and spin down; denature proteins at  for ; briefly spin down.
5 µl
20 µl
100 °C
?. [Endoglycosidase H (Endo H) digestion assay]
PREPARE Novex® 10% Tris-glycine gel with adapter. For 1 gel prepare  running buffer:  10X Tris-glycine-SDS running buffer (Boston BioProducts) +  diH2O.
500 mL
50 mL
450 mL
No need to recycle running buffer.
?. [Endoglycosidase H (Endo H) digestion assay]
Load  of the protein ladder and the entire reaction volume of the +/- Endo H samples – first all -Endo H samples followed by all +Endo H samples.
8 µl
?. [Endoglycosidase H (Endo H) digestion assay]
Run the gel:
?. [Endoglycosidase H (Endo H) digestion assay]
Wet electrophoretic transfer onto a PVDF membrane (Bio-Rad) with 10X Tris-glycine transfer buffer (Boston BioProducts):  diH2O +  10X Tris-glycine transfer buffer +  MeOH [20% (v/v)].
700 mL
100 mL
200 mL
?. [Endoglycosidase H (Endo H) digestion assay]
Continue with immunoblotting using the Odyssey infrared imaging system (LI-COR Biosciences).
?. [Endoglycosidase H (Endo H) digestion assay]
Prepare a plate for the BCA test; each condition in triplicate.
?. [Endoglycosidase H (Endo H) digestion assay]
Label Eppis and chill them on wet ice.
?. [Endoglycosidase H (Endo H) digestion assay]
Get dry ice.
?. [Endoglycosidase H (Endo H) digestion assay]
Refer to the spreadsheet for mixing an appropriate volume of the cell lysate (- protein) with diH2O for a total volume of ; from this point onwards samples can be kept at .
30 µg
40 µg
15.3 µl
-20 Room temperature
?. [Endoglycosidase H (Endo H) digestion assay]
Add  of the glycoprotein denaturing buffer for a total reaction volume of .
1.7 µl
17 µl
?. [Endoglycosidase H (Endo H) digestion assay]
Boil at  for . In the meantime transfer the cell lysate stock to .
100 °C
-80 °C
Make sure that the lids of the Eppis are tightly closed!
?. [Endoglycosidase H (Endo H) digestion assay]
Briefly vortex and spin down.
?. [Endoglycosidase H (Endo H) digestion assay]
In the meantime PREPARE the +/- Endo MMs:MM-1 (+ Endo H): ( Endo H +  10X G5 buffer)/rxn; can be kept at .MM-2 (- Endo H): ( diH2O +  10X G5 buffer)/rxn.
1 µl
2 µl
-80 Room temperature
1 µl
2 µl
?. [Endoglycosidase H (Endo H) digestion assay]
Spin down briefly.
?. [Endoglycosidase H (Endo H) digestion assay]
Incubate at  for  (minimum incubation time).
37 °C
?. [Endoglycosidase H (Endo H) digestion assay]
Spin down samples briefly; add  of MM-1 or -2, briefly vortex and spin down.
3 µl
?. [Endoglycosidase H (Endo H) digestion assay]
Store samples at  or  if the gel is to be run on another day.
-80 °C
-20 °C
?. [Endoglycosidase H (Endo H) digestion assay]
Run at 100 V first for  – until the running front is completely condensed by means of the stacking gel.
?. [Endoglycosidase H (Endo H) digestion assay]
Then increase voltage to 120 V and set timer to .Run the gel until the 37 kDa protein ladder band is approximately - above the lower end of the gel cassette; check gel regularly after ; don’t run too far!!.
1
1.5
?. [Endoglycosidase H (Endo H) digestion assay]
Prepare  transfer buffer and keep in  cold room; can be recycled 3-5x.
2 L
4 °C
?. [Endoglycosidase H (Endo H) digestion assay]
Transfer for  at 60 V and  or for  at 65 V in the case of two simultaneous transfers.
4 °C
?. [Endoglycosidase H (Endo H) digestion assay]
Antibodies:Primary antibodySecondary antibodyRb anti-GCase1:500Gt anti-rb IgG, DyL 8001:5,000Rb anti-nicastrin1:500Ms anti-GAPDH1:3,000Gt anti-ms IgG, DyL 6801:10,000
Primary antibodySecondary antibodyRb anti-GCase1:500Gt anti-rb IgG, DyL 8001:5,000Rb anti-nicastrin1:500Ms anti-GAPDH1:3,000Gt anti-ms IgG, DyL 6801:10,000