Endoglycosidase H (Endo H) digestion assay

PREPARE:

Make sure the lysis buffer (Table 1) is chilled.

Cool down ultracentrifuge to 4°C. Set parameters: TLA-100 rotor, 100000x g,0h 0m 0s, 0h 30m 0s, 4°C; apply vacuum.

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 4On ice from this point onwards. The protease inhibitor cocktail can be briefly thawed using the 37°C heat block. Keep at 37Room temperature before re-freezing.

Get a large box and fill to approximately 2/3 with wet ice for the ice/water slurry (see below).

Get samples and keep on dry ice.

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.

Resuspend the cell pellet to a homogenous (!) solution by trituration and keep on wet ice.

Prepare the ice/water slurry: add cold water to approximately half of the height of the ice layer.

Incubate samples for 0h 20m 0s in the ice/water slurry.

In the meantime PREPARE the following:

Label ultracentrifuge tubes on the side. Use P200 tip holder as a rack for the ultracentrifuge tubes.

Prepare dry ice/EtOH slurry approximately 0h 10m 0s before the 0h 20m 0s incubation on the ice/water slurry is completed:

• Fill small box to 2/3 with EtOH (190 proof, 95%).
• Add dry ice until saturation (dry ice doesn’t resolve quickly anymore; temperature of-80°C has stabilized).

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 0h 1m 0s.

Directly transfer Eppis onto a heat block and incubate at 37°C for exactly (!) 0h 1m 0s (no longer!).

Repeat the described freeze/thaw cycle once → two freeze/thaw cycles (-80°C/37°C) in total.

Keep samples on wet ice afterwards.

Get TLA-100 rotor (Beckman Coulter) and keep on wet ice, including the lid. TLA-100 rotor usually kept in the 4°C cold room.

Transfer the cell lysate from the Eppis into the labeled ultracentrifuge tubes (thickwall polycarbonate tubes) and place them in the TLA-100 rotor 4On ice; keep the empty Eppis for a later step in case the labeling is sufficient and hasn’t been removed by the EtOH.

Place TLA-100 rotor into the ultracentrifuge; make sure it’s correctly placed (!); re-apply vacuum, make sure that the parameters are set correctly (100000x g,0h 0m 0s for 0h 30m 0s at 4°C) 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 4°C.

During the ultracentrifugation PREPARE the following:

Prepare a plate for the BCA test; each condition in triplicate.

Label Eppis and chill them on wet ice.

Get dry ice.

Get TLA-100 rotor and keep on wet ice.

Remove ultracentrifuge tubes and transfer the supernatant to a new, correctly labeled and chilled Eppi; keep Eppis on wet ice.

A small cell pellet should be visible; transfer to the appropriate old, correctly labeled Eppi and keep on dry ice.

For the BCA test either add 1µL of the cell lysate to 24µL water or dilute 2µL of the cell lysate in a 1:1 ratio with 2µL water before adding 1µL of the 1:2 dilution to 24µL water; in the latter case don’t forget to change the dilution factor on the Endo H spreadsheet to 50!.

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 -80°C (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 4°C cold room.

During the incubation for the BCA test at 37°C for 0h 26m 0s roughly label 2 Eppis for each condition (→ +/- Endo H).

Thaw 10X glycoprotein denaturing buffer and 10X G5 buffer at 4Room temperature (don’t vortex denaturing buffer as this will create a lot of small bubbles!).

After BCA test use Chee-Yeun’s Endo H spreadsheet to calculate the protein concentration.

PREPARE: thaw Endo H (stored at -20°C) on wet ice.

From this point onwards all in duplicate (→ +/- Endo H):

Refer to the spreadsheet for mixing an appropriate volume of the cell lysate (30µg-40µg protein) with diH₂O for a total volume of 15.3µL; from this point onwards samples can be kept at -20Room temperature.

Add 1.7µL of the glycoprotein denaturing buffer for a total reaction volume of 17µL.

Briefly vortex and spin down.

Boil at 100°C for 0h 10m 0s. In the meantime transfer the cell lysate stock to -80°C.

In the meantime PREPARE the +/- Endo MMs:

MM-1 (+ Endo H): (1µL Endo H + 2µL 10X G5 buffer)/rxn; can be kept at -80Room temperature.

MM-2 (- Endo H): (1µL diH2O ₂O + 2µL 10X G5 buffer)/rxn.

Spin down samples briefly; add 3µL of MM-1 or -2, briefly vortex and spin down.

Incubate at 37°C for 2h 0m 0s (minimum incubation time).

Spin down briefly.

Store samples at -80°C or -20°C if the gel is to be run on another day.

Thaw sample buffer (“5X SB”, kept at -20°C; Table 2) and protein ladder at -20Room temperature.

In case +/- Endo H samples have been frozen down, thaw at -20Room temperature and briefly spin down.

Add 5µL 5X SB to each 20µL rxn; briefly vortex and spin down; denature proteins at 100°C for 0h 10m 0s; briefly spin down.

PREPARE Novex® 10% Tris-glycine gel with adapter. For 1 gel prepare 500mL running buffer: 50mL 10X Tris-glycine-SDS running buffer (Boston BioProducts) + 450mL diH₂O.

Load 8µL 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.

Run the gel:

Run at 100 V first for 0h 20m 0s – until the running front is completely condensed by means of the stacking gel.

Then increase voltage to 120 V and set timer to 2h 0m 0s.

• Run the gel until the 37 kDa protein ladder band is approximately 1cm-1.5cm above the lower end of the gel cassette; check gel regularly after 1h 0m 0s; don’t run too far!!.

Wet electrophoretic transfer onto a PVDF membrane (Bio-Rad) with 10X Tris-glycine transfer buffer (Boston BioProducts): 700mL diH₂O + 100mL 10X Tris-glycine transfer buffer + 200mL MeOH [20% (v/v)].

Prepare 2L transfer buffer and keep in 4°C cold room; can be recycled 3-5x.

Transfer for 2h 0m 0s at 60 V and 4°C or for 2h 30m 0s at 65 V in the case of two simultaneous transfers.

Continue with immunoblotting using the Odyssey infrared imaging system (LI-COR Biosciences).

Antibodies:

A	B	C	D
Primary antibody		Secondary antibody
Rb anti-GCase	1:500	Gt anti-rb IgG, DyL 800	1:5,000
Rb anti-nicastrin	1:500
Ms anti-GAPDH	1:3,000	Gt anti-ms IgG, DyL 680	1:10,000