Jun 19, 2025
Sucrose Density Gradient Co-flotation of PPM1H and Rab10
- Ayan Adhikari1,
- Suzanne R. Pfeffer1
- 1Stanford University School of Medicine, Stanford, California USA 94305-5307
External link: https://doi.org/10.1016/j.jbc.2025.110679
Protocol Citation: Ayan Adhikari, Suzanne R. Pfeffer 2025. Sucrose Density Gradient Co-flotation of PPM1H and Rab10. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g79o2kvwz/v1
Manuscript citation:
Adhikari A, Tripathi A, Chiang CY, Sherpa P, Pfeffer SR (2025) Allosteric regulation of the Golgi-localized PPM1H phosphatase by Rab GTPases modulates LRRK2 substrate dephosphorylation in Parkinson’s disease. The Journal of Biological Chemistry 301(10). doi: 10.1016/j.jbc.2025.110679
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: June 19, 2025
Last Modified: June 19, 2025
Protocol Integer ID: 220555
Keywords: ASAPCRN, ppm1h phosphatase to unphosphorylated rab10, unphosphorylated rab10, liposome, ppm1h phosphatase, diameter liposome, rab10 sucrose, potential binding of mneon, bound protein, protein, flotation assay, rab10, recombinant mneon
Funders Acknowledgements:
Aligning Science Across Parkinson's
Grant ID: ASAP-000463
Abstract
Sucrose gradient co-flotation assays are employed to assess the potential binding of mNeon-PPM1H phosphatase to unphosphorylated Rab10. Recombinant mNeon-PPM1H and Rab10 are incubated with 50 nm-diameter liposomes for 30 min at 30°C. The mixture is layered beneath a discontinuous sucrose gradient and then subjected to ultracentrifugation to resolve liposome-bound proteins. Following centrifugation, fractions are analyzed by immunoblotting to detect the distributions of both mNeon-PPM1H and Rab10.
Materials
1. Eppendorf LoBind tubes, 0.5mL Catalog No. 0030108434
2. Eppendorf LoRetention tips, 0.1-10uL Catalog No. 022493018
2. Bacterially expressed, full length untagged mNeon PPM1H and full length untagged Rab10 Q68L.
3. Molecular grade sucrose (Millipore #573113)
4. Ultra-clear centrifuge tubes (Beckman Coulter #344090)
5. Freshly prepared 50nm liposomes (produced by using a mini hand extruder (Avanti # 610000 1EA).
Troubleshooting
Safety warnings
Freshly prepared liposomes work best
Purification of mNeon PPM1H and Rab10
mNeon PPM1H and Rab10 are expressed in Escherichia coli BL21 (DE3) pLysS Rosetta cells. Protein purification is performed using Cobalt-affinity chromatography followed by size-exclusion chromatography. Details are available at:
For Rab10 purification, 20 μM GTP is included in all buffers to stabilize the GTP-bound conformation. The Rab10 Q68L mutant is used to enrich for the GTP-bound state.
Preparation of Small Unilamellar Vesicles (SUVs)
Small (50nm) unilamellar vesicles (SUVs) are formed by extrusion using (18:0-20:4)PC, (18:0-20:4)PI, (18:0-18:2)PS, (18:1) plus PI(4)P, and cholesterol at a molar ratio of 78:7:5:1:9 (Avanti Polar Lipids; ThermoFisher) to reproduce the composition of the trans Golgi network. Details can be found at:
Sucrose Density Gradient Co-flotation
50 nm liposomes (1.5 mM) are incubated with mNeon PPM1H (1µM final) and Rab10 (3µM final) for 30 minutes at 30°C in a total volume of 33 μl of HKM buffer (20 mM HEPES pH-7.5, 150 mM potassium acetate and 1 mM magnesium chloride) containing 20µM GTP (HKM/GTP).
167 μl of 60% w/v sucrose (in HKM/GTP) is then added and mixed well with the above-mentioned suspension to adjust it to 50% sucrose final, 1X HKM/GTP.
This high sucrose suspension is carefully overlaid with 200 μl 25% w/v sucrose in HKM/GTP followed by 100 μl of HKM/GTP buffer.
The sample is centrifuged at 45000 RPM for 3 hours using an SW55Ti swinging bucket rotor.
Ten 50 μl fractions are manually collected from the top of the sucrose gradient by using a micropipette held against the side of the tube.
The fractions are then analyzed by Western blotting to detect mNeon PPM1H and Rab10 using anti-rabbit PPM1H antibody and anti-mouse Rab10 antibody.
Untitled section
This immunoblot shows the sucrose gradient flotation profile of PPM1H in the absence (top) or presence (bottom) of small 50nm liposomes. Top of the gradient is shown at left; mass of molecular weight marker in kDa is also shown at left. Reprinted from https://doi.org/10.1073/pnas.2315171120.