Aug 29, 2024
ATG9A-vesicle isolation protocol
- 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna (AT)
Protocol Citation: Daniel Bernklau, Justyna Sawa-Makarska, Julia Romanov 2024. ATG9A-vesicle isolation protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpzok8lzp/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: July 04, 2024
Last Modified: August 29, 2024
Protocol Integer ID: 103561
Keywords: ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP)
Grant ID: ASAP-000350
Marie Skłodowska-Curie MSCA Postdoctoral fellowship
Grant ID: 101062916
Abstract
This protocol details about the ATG9-vesicle isolation.
Materials
Sodium-Orthovanadate (Applichem, prepared as 100 millimolar (mM) in MQ, frozen)
b-Glycerophosphate (Sigma, prepared as 1 Molarity (M) in MQ, frozen)
NaF (Sigma, prepared as 500 millimolar (mM) in MQ, frozen)
Complete EDTA free protease inhibitor tablets, Sigma 5056489001 (prepared as 50x stock 1 tbl in 1 ml H2O)
Anti-FLAG M2 Affinity Gel from Sigma A2220-1ml
D(+)-Saccharose (sucrose) from Roth 4661.1
FLAG Peptide from Sigma F32920-4MG
Dissolved at 4 mg /ml in SEC buffer ( 25 millimolar (mM) NaCl, 1 millimolar (mM) DTT), aliquoted, kept at -20 °C
3 mL Luer Lock HENKE-JECT syringe
Braun Sterican 26 G x 1‘‘ Gr. 18 needle
MilliQ water (filtered additionally through a 0.2μm membrane)
All buffers used were filtered prior to use
0.2 μm celluloseacetate syringe filter from Chromafil CA-20/25 (S)
| A | B | C | D | E | F | |
| Vesicle Isolation Buffer | Elution Base Buffer | |||||
| Reagent | Stock conc | Final conc | Vol for 20ml | Final Conc | Vol for 20ml | |
| HEPES pH7.5 | 1M | 20mM | 400μl | 20mM | 400μl | |
| NaCl | 5M | 150mM | 600μl | 150mM | 600μl | |
| Sucrose | 1M | 250mM | 5ml | - | - | |
| Complete EDTA free protease inhibitors (PI) | 50x | 1x | 400μl | 1x | 400μl | |
| Beta-Glycerophosphate | 1M | 20mM | 400μl | 20mM | 400μl | |
| Sodium-Orhovanadate | 100mM | 1mM | 200μl | 1mM | 200μl | |
| NaF | 500mM | 1mM | 40μl | 1mM | 40μl | |
| EDTA pH8.0 | 0.5M | 1mM | 40μl | - | - | |
| In MilliQ H2O | ||||||
| Sterile filtered, precooled | ||||||
Before start
Hap1 cells were previously CRISPR engineered in our lab to contain ATG9A endogenously tagged on the
C-terminus with mEGFP-3C-FLAG.
Preparation of starting material (Hap1 ATG9A-mEGFP-3C-FLAG cell pellets)
Preparation of starting material (Hap1 ATG9A-mEGFP-3C-FLAG cell pellets)
Grow and expand cells in IMDM medium supplied with 10%
FCS and Pen/Strep until the required number of cells is reached.
Prep size: For one preparation of vesicles the required amount is
100 x 10^6 to 150 x 10^6 cells which is equivalent to 4 - 5 x 15 cm dishes of confluent
cells.
Detach cells with trypsin, count and aliquot to prep size (see above), wash with cold PBS, spin
down 200 rcf, 4°C , remove supernatant, flash freeze the cell pellets and keep
at -80 °C until use.
2w
Day1: Cell lysis and binding to Flag beads
Day1: Cell lysis and binding to Flag beads
1h
1h
Equilibrate Flag beads
Always pellet the beads with 4000 rcf, 4°C cooled 5415R centrifuge for 3’.
Take 70 µL of FLAG bead slurry per prep.
Wash 3x with MilliQ water
Wash 3x with Vesicle Isolation Buffer.
Prepare a 1 : 1 slurry in Vesicle isolation Buffer
Cell lysis
Always work On ice
">> sample name" means: take a sample for Western Blot e.g. 10 µl sample + 2 µl 6xProtein loading dye
Thaw cells On ice
Resuspend the pellet in 1665 µL of Vesicle Isolation Buffer and transfer to a
fresh 2ml Eppi.
Incubate On ice for 00:20:00
20m
Lyse the cells by pushing them 30 times (up and down) through a 26 x g needle
10m
Let them chill for 00:10:00 On ice
10m
Repeat the lysis step (push 30 times up and down through a 26 x g needle)
10m
Pellet the cell debris and nuclei (200 rcf, 4°C, 00:06:00 )
6m
Transfer the supernatant to a fresh 2 ml Eppi (>> Sample Supernatant)
Add 70 µL of equilibrated FLAG beads (slurry).
Incubate Overnight at 4 °C (on the roller in the cold room in a 50ml Falcon tube)
10m
Day2 Elution of ATG9A vesicles
Day2 Elution of ATG9A vesicles
3h 15m
3h 15m
Pellet the beads (in a 5415R centrifuge) at 200 rcf, 00:03:00 and remove the supernatant (>> Sample Unbound).
3m
Resuspend the beads in 1 mL of Vesicle Isolation Buffer and transfer to a fresh 2ml Eppi (+665 µL Vesicle Isolation Buffer already pre-pipetted in the Eppi).
Incubate 00:01:00 rolling at 4 °C or on ice with occasionally inverting the Eppi carefully
1m
Pellet the beads at 2000 rcf, 00:03:00 and remove the supernatant
3m
Resuspend the beads in 1 mL of Vesicle Isolation Buffer and transfer into a fresh 2 ml LowBind tube
Add 665 µL of Elution Base Buffer very slowly to the tube (decreases the sucrose concentration;
now be even more careful than before!)
Incubate 00:01:00 rolling at 4 °C or on ice with occasionally inverting the tube carefully
1m
Pellet the beads at 2000 rcf, 00:03:00 and remove the supernatant
3m
Resuspend the beads in 1 mL of Elution Base Buffer and transfer into a fresh 2 ml LowBind tube665 µL Elution Base Buffer already in pre-pipetted in the tube).
Incubate 00:01:00 rolling at 4 °C or on ice with occasionally inverting the tube carefully
1m
Pellet the beads at 2000 rcf, 00:03:00 and remove the supernatant.
3m
Resuspend the beads in 666 µL of Elution Base Buffer and transfer to a fresh 1.5 ml LowBind tube (>> Sample Elution Input).
Add 16.65 µL of 4 mg /ml FLAG peptide
Incubate for 02:45:00 at 4°C rolling
2h 45m
Pellet the beads at 2000 rcf, 00:03:00 and transfer the supernatant to a fresh LowBind tube (>> Sample Elution)
This is your vesicle prep! Handle with care.
3m
Resuspend the beads again in 666 µL of Elution Base Buffer (>>Sample Beads after Elution)