Aug 24, 2022
Preparation and imaging of enriched Golgi from GolgiTAG-IP using Transmission Electron Microscopy
- Rotimi Fasimoye1,
- Alan Prescott2,
- Dario R Alessi1
- 1Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK;
- 2Dundee Imaging Facility, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK
- asap
External link: https://doi.org/10.1073/pnas.2219953120
Protocol Citation: Rotimi Fasimoye, Alan Prescott, Dario R Alessi 2022. Preparation and imaging of enriched Golgi from GolgiTAG-IP using Transmission Electron Microscopy. protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9y9nqg3e/v1
Manuscript citation:
Fasimoye R, Dong W, Nirujogi RS, Rawat ES, Iguchi M, Nyame K, Phung TK, Bagnoli E, Prescott AR, Alessi DR, Abu-Remaileh M, Golgi-IP, a tool for multimodal analysis of Golgi molecular content. Proceedings of the National Academy of Sciences of the United States of America 120(20). doi: 10.1073/pnas.2219953120
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: August 22, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 69022
Keywords: Enriched Golgi imaging, GolgiTAG-IP, Transmission Electron Microscopy, ASAPCRN, imaging of enriched golgi, cells by golgitag immunoprecipitation, golgitag immunoprecipitation, using transmission electron microscopy transmission electron microscopy, transmission electron microscopy transmission electron microscopy, golgi, organelle, enriched golgi, structure of organelle, using various organelle, various organelle, golgitag, intact cell, cell, fixed intact cell, imaging
Funders Acknowledgements:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-000463
UK Medical Research Council
Grant ID: MC_UU_00018/1
Abstract
Transmission electron microscopy (TEM) is a tool used to image, in good resolution, the structure of organelles within the cell. Available protocols are designed to image structures within fixed intact cells. Here, we described a protocol where Golgi, isolated from cells by GolgiTAG immunoprecipitation (IP) (as described in ddx.doi.org/10.17504/protocols.io.6qpvrdjrogmk/v1), can be prepared and imaged using TEM. This protocol can also be used to image any organelles isolated using various organelle-IP protocols that are available.
Attachments
ivgnbgrdf.docx
804KB
Materials
Materials:
- ParaformaldehydeMerck MilliporeSigma (Sigma-Aldrich)Catalog #158127
- Glutaraldehyde EM Grade 25%Merck MilliporeSigma (Sigma-Aldrich)Catalog #G5882-50ML
- Sodium cacodylate trihydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #C0250
- Osmium tetroxide (Agar. Catalog # R1023)
- Sodium ferrocyanide decahydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #13425
- Tannic acid (BDH. Catalog # 30337)
- Uranyl acetate (Agar. Catalog # R1260A)
- Durcupan™ ACMMerck MilliporeSigma (Sigma-Aldrich)Catalog #44611
- Lead(II) citrate tribasic trihydrateMerck MilliporeSigma (Sigma-Aldrich)Catalog #15326
- Sodium citrate monobasicMerck MilliporeSigma (Sigma-Aldrich)Catalog #71497
- Sodium hydroxide (NaOH)Merck MilliporeSigma (Sigma-Aldrich)Catalog #S5881
- Potassium chloride (KCl) (VWR. Catalog# 267264)
- Potassium phosphate monobasicMerck MilliporeSigma (Sigma-Aldrich)Catalog #P5379
- Calcium chloride (Calbiochem. Catalog# 208291)
- (R)-( )-Propylene oxideMerck MilliporeSigma (Sigma-Aldrich)Catalog #540048
- Ethyl alcohol, 200 proof, anhydrous, ≥99.5%Merck MilliporeSigma (Sigma-Aldrich)Catalog #459836
Buffer:
- KPBS (136 millimolar (mM) KCl, 10 millimolar (mM) KH2PO4. Adjust to pH 7.25 with KOH).
- 0.4 Molarity (M) sodium cacodylate buffer (17.12 g sodium cacodylate dissolved in water. Adjust to pH7.4 with HCl).
- Reynold lead citrate (1.33 g lead citrate, 1.76 g sodium citrate; 8 mL 1N NaOH in 50 mL water).
Equipment:
- Pierce™ Anti-HA Magnetic BeadsThermo FisherCatalog #88837
- DynaMag™- Spin MagnetThermo FisherCatalog #12320D
- Microcentrifuge with thermostat (VWR Micro Star 17R. S/N 42209232)
- Scalpel
- P1000 pipette tips
- Pasteur pipette
- Oven (TAAB. I064)
- Leica UCT ultramicrotome (Leica)
- Clear glass with solid top black phenolic 14B rubber lined capVWR International (Avantor)Catalog #215-3571
- JEOL 1200EX TEM using SIS III camera
Troubleshooting
Method
2d 5h 4m
Perform GolgiTAG-IP as previously described in dx.doi.org/10.17504/protocols.io.6qpvrdjrogmk/v1
After last wash of beads with KPBS, fix the beads in 1 mL solution containing 4% (w/v) paraformaldehyde and 2.5% (v/v) glutaraldehyde in 0.1 Molarity (M) sodium cacodylate buffer dilute in water for 01:00:00 at Room temperature .
1h
Pellet beads with magnet DynaMagTM Spin Magnet (or equivalent) and remove the supernatant.
Wash beads pellet three times in 1 mL 0.1 Molarity (M) sodium cacodylate buffer, pelleting the beads with magnet after each wash.
After third wash, spin down beads in tabletop centrifuge at 1000 x g for 00:01:00 . Pellet should be tightly packed.
1m
Use a needle to gently dislodge the pellet and use a Pasteur pipette to remove the pellet to a clean glass vial. (Note: All steps are carried out in a glass vial).
To ensure rapid dehydration and embedding, cut pellet with scalpel into approximately 1mm3 pieces, then post-fix in 1 mL 1% (w/v) Osmium tetroxide with 1.5% (w/v) sodium ferrocyanide in 0.1 Molarity (M) sodium cacodylate buffer for 01:00:00 at Room temperature .
1h
Wash pellets three times in 0.1 Molarity (M) sodium cacodylate buffer, by adding the buffer, allowing the pellets to settle and gently taking out supernatant with P1000 tip.
Wash pellets three times in 0.1 Molarity (M) sodium cacodylate buffer for 00:01:00 (1/3).
1m
Wash pellets three times in 0.1 Molarity (M) sodium cacodylate buffer for 00:01:00 (2/3).
1m
Wash pellets three times in 0.1 Molarity (M) sodium cacodylate buffer for 00:01:00 (3/3).
1m
After the third wash, wash with water three times (as in step 8), then resuspend in 1% w/v tannic acid and 1% w/v uranyl acetate diluted in water for 01:00:00 . Remove supernatant.
1h
Dehydrate pellet through alcohol series:
50% ethanol for 00:10:00
70% ethanol for 00:10:00
80% ethanol for 00:10:00
90% ethanol for 00:10:00
95% ethanol for 00:10:00
then twice in 100% ethanol for 00:10:00 (1/2)
100% ethanol for 00:10:00 (2/2)
Note
Use a P1000 pippete to gently remove ethanol after each series.
1h 10m
Further dehydrate in 100% propylene oxide twice for 10 min.
Further dehydrate in 100% propylene oxide twice for 00:10:00 (1/2).
10m
Further dehydrate in 100% propylene oxide twice for 00:10:00 (2/2).
10m
Resuspend pellet into 50% v/v propylene oxide and 50% v/v Durcupan resin Overnight .
10m
Open glass vial to allow propylene oxide to evaporate (This will take approximately 01:00:00 ).
1h
To embed pellet in resin, resuspend in 100% Durcupan resin.
Embed polymerise resin in an oven at 60 °C for 48:00:00 .
2d
Cut sections of polymerised embedded resin with Leica UCT ultramicrotome.
Note
Sections should be 70-100nm thick
Stain sections with 3% aqueous uranyl acetate for 00:20:00 .
20m
Stain with Reynold lead citrate for 00:20:00 .
20m
Image stained sections on JEOL 1200EX TEM using SIS III camera.
Note
Note: Due to the cutting and many centrifugation steps, it is expected that the magnetic particles dissociate from the plastic bead core.)