Sep 23, 2023

Public workspacePurification of NDP52 (untagged)

DOI
dx.doi.org/10.17504/protocols.io.36wgq35xklk5/v1
  • 1Sascha Martens lab, University of Vienna, Max Perutz Labs - Vienna
Elias Adriaenssens
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DOI: dx.doi.org/10.17504/protocols.io.36wgq35xklk5/v1
Protocol Citation: Elias Adriaenssens 2023. Purification of NDP52 (untagged). protocols.io https://dx.doi.org/10.17504/protocols.io.36wgq35xklk5/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 07, 2023
Last Modified: May 31, 2024
Protocol Integer ID: 84642
Keywords: Purification of NDP52 (untagged), 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 describes purification of NDP52 (untagged).
Attachments
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775-1961.pdf
56KB
Materials
Materials

  • Human NDP52 cDNA in pGST2 vector with an N-terminal GST tag (RRID:Addgene #187828)
  • IPTG
  • SORVAL RC6+ centrifuge with an F21S8x50Y rotor (Thermo Scientific)
  • Glutathione Sepharose 4B beads (GE Healthcare)
  • 30 kDa cut-off Amicon filter (Merck Millipore)
  • Superdex 200 Increase 10/300 GL column (Cytiva)

Lysis buffer
AB
Tris-HCl pH 7.450 mM
NaCl300 mM
DTT1 mM
MgCl22 mM
β-mercaptoethanol2 mM
cOmplete EDTA-free protease inhibitors (Roche)
DNase (Sigma)
Wash buffer
AB
Tris-HCl pH 7.450 mM
NaCl300 mM
DTT1 mM
High salt wash buffer
AB
Tris-HCl pH 7.450 mM
NaCl700 mM
DTT1 mM
SEC buffer
AB
Tris-HCl pH 7.425 mM
NaCl150 mM
DTT1 mM
Purification of NDP52 (untagged)
Purification of NDP52 (untagged)
16h
16h
The human NDP52 cDNA into a pGST2 vector with an N-terminal GST tag followed by a TEV cleavage site is available from Addgene (RRID: Addgene #187828).
After the transformation of the pGST2 vector encoding GST-TEV-NDP52 in E. coli Rosetta pLySS cells, grow cells in 2xTY medium at Temperature37 °C until an OD600 of 0.4 and then continued at Temperature18 °C .
Once the cells reached an OD600 of 0.8, induce protein expression with Concentration50 micromolar (µM) IPTG for Duration16:00:00 at Temperature18 °C .
16h
Collect cells by centrifugation and resuspend in lysis buffer.
Centrifigation
Sonicate cell lysates.
Sonicate cell lysates for Duration00:00:30 . (1/2)

30s
Sonicate cell lysates for Duration00:00:30 . (1/2)
30s
Clear lysates by centrifugation at Centrifigation18000 rpm, 4°C, 00:45:00 in a SORVAL RC6+ centrifuge with an F21S8x50Y rotor (Thermo Scientific).
45m
Centrifigation
Collect the supernatant and incubate with preequilibrated Glutathione Sepharose 4B beads (GE Healthcare) for Duration02:00:00 at Temperature4 °C with gentle shaking to bind GST-NDP52.
2h
Incubation
Centrifuge the samples to pellet the beads and remove the unbound lysate.
Centrifigation
Wash the beads.
Wash
Wash the beads twice with wash buffer.
Wash the beads with high salt wash buffer.
Again, wash the beads twice with wash buffer.
Incubate beads DurationOvernight with TEV protease at Temperature4 °C .

Incubation
Overnight
After the GST tag was cleaved off, filter the protein through a Thikness0.45 µm syringe filter, concentrated using a 30 kDa cut-off Amicon filter (Merck Millipore), and load onto a pre-equilibrated Superdex 200 Increase 10/300 GL column (Cytiva).
Elute proteins with SEC buffer.
Analyze fractions by SDS-PAGE and Coomassie staining.
Analyze
Pool fractions containing purified NDP52.
After concentrating the purified protein, aliquot the protein and snap-freeze in liquid nitrogen. Store the proteins at Temperature-80 °C .
Temperature