Aug 23, 2022
Immunofluorescence imaging of cells expressing GolgiTAG (TMEM115-3HA) or HA-tagged non-Golgi annotated proteins
- Rotimi Fasimoye1,
- Dario R Alessi1
- 1Medical Research Council Protein Phosphorylation and Ubiquitylation Unit, School of Life Sciences, University of Dundee, Dow Street, Dundee DD1 5EH, UK
External link: https://doi.org/10.1073/pnas.2219953120
Protocol Citation: Rotimi Fasimoye, Dario R Alessi 2022. Immunofluorescence imaging of cells expressing GolgiTAG (TMEM115-3HA) or HA-tagged non-Golgi annotated proteins. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g74qpkgwz/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 16, 2022
Last Modified: May 31, 2024
Protocol Integer ID: 68707
Keywords: GolgiTAG, Non-Golgi annotated proteins, Immunofluorescence, ASAPCRN
Funders Acknowledgement:
Aligning Science Across Parkinson’s (ASAP) initiative
Grant ID: ASAP-000463
Abstract
Immunofluorescent (IF) microscopy is a powerful tool used in cellular and molecular biology to monitor the subcellular localisation of proteins. By combining the advantages of immunostaining and confocal light microscope, IF microscopy can be used to assess the colocalization of two or more proteins within the cell. Here, we describe a method that can be used to verify the correct localisation of HA-tagged Golgi proteins (like TMEM115-3HA, or GolgiTAG), by assessing their colocalisation with known Golgi markers such as GM130, GOLGIN97 and ACBD3. Furthermore, this method can be used to investigate whether HA-tagged, non-Golgi annotated proteins transiently expressed in cells localise at the Golgi, by assessing their colocalisation with ACBD3.
Attachments
ius4bgrdf.docx
291KB
Materials
Cell lines:
- HEK293 (ATCC Catalog number CRL-1573, RRID:CVCL_0045)
- HeLa (ATCC Catalog number CCL-2, RRID:CVCL_0030)
Plasmids:
See Table 1. All plasmids are available from the MRC PPU Reagents and Services (https://mrcppureagents.dundee.ac.uk).
(Note we purify plasmids using a QIAGEN HiSpeed® Plasmid Maxi kit [Lot# 166034460] following the manufacturer’s instructions and ensure sterile reagents are used to avoid contamination).
Table 1: List of Plasmids. All plasmids are available from the MRC PPU Reagents and Services (https://mrcppureagents.dundee.ac.uk).
| A | B | C | |
| Plasmids | Gene expressed | Plasmid Backbone | |
| DU72430 | KIAA2013-HA | pcDNA5D | |
| DU72445 | TM9SF1-HA | pcDNA5D | |
| DU72446 | DIPK1A-HA | pcDNA5D | |
| DU72447 | C5orf22-HA | pcDNA5D | |
| DU72466 | SLC39A9-HA | pcDNA5D | |
| DU72705 | TMEM219-HA | pcDNA5D | |
| DU72706 | ABHD13-HA | pcDNA5D | |
| DU72709 | CCDC126-HA | pcDNA5D |
Antibodies
- See Tables 2 and 3
ACBD3 (clone 2G2)Sigma – AldrichCatalog #WH0064746M1
Golgin-97 (D8P2K) Rabbit mAbCell Signaling TechnologyCatalog #13192
GM130 (D6B1) XP® Rabbit mAbCell Signaling TechnologyCatalog #12480
HASigmaCatalog #11867423001
Table 2: List of primary antibodies used for immunofluorescence staining.
| A | B | C | D | E | |
| Antibody | Company | Cat. Number (RRID) | Host species | Dilution for IF | |
| ACBD3 (clone 2G2) | Sigma | WH0064746M1 (RRID:AB_2220068) | Mouse | 1:1000 | |
| GOLGIN97 | Cell Signalling Technology | 13192 (RRID:AB_2798144) | Rabbit | 1:100 | |
| GM130 | Cell Signalling Technology | 12480 (RRID:AB_2797933) | Rabbit | 1:3000 | |
| GCC185 (serum) | Pfeffer’s lab | (PMID: 31663853) | Rabbit | 1:500 | |
| HA | Sigma | 11867423001 (RRID:AB_390918) | Rat | 1:1000 |
Anti-RabbitInvitrogen - Thermo FisherCatalog #A21206
Anti-RatInvitrogen - Thermo FisherCatalog #A21209
anti-MouseInvitrogen - Thermo FisherCatalog #A21202
Table 3: List of fluorophore-conjugated secondary antibodies. All secondary antibodies are used at a 1:500 dilution.
| A | B | C | D | E | |
| Antibody | Conjugated Fluorophore | Company | Cat. Number (RRID) | Host Species | |
| anti-Rat | Alexa 594 | Invitrogen | A21209 (RRID:AB_2535795) | Donkey | |
| anti-Rabbit | Alexa 488 | Invitrogen | A21206 (RRID:AB_2535792) | Donkey | |
| anti-Mouse | Alexa 488 | Invitrogen | A21202 (RRID:AB_141607) | Donkey |
Media and Reagents:
- Growth Media:
| A | B | |
| Dulbecco’s Modified Eagle’s Medium (DMEM) (GIBCO 11960-085) | ||
| Foetal Bovine Serum (FBS) (Sigma F7524 Batch# BCBW6817) | 10% (v/v) | |
| L-Glutamine (GIBCO 25030024) | 1% | |
| Penicillin-Streptomycin (GIBCO 15140122) | 1% |
DMEM, high glucose, no glutamineThermo FisherCatalog #11960085
Fetal Bovine SerumSigma AldrichCatalog #F7524
L-Glutamine (200mM)Thermo Fisher ScientificCatalog #25030024
Penicillin-StreptomycinGibco - Thermo FisherCatalog #15140122
- Transfection media:
- Opti-MEM™ I Reduced Serum MediumGibco - Thermo FischerCatalog #31985062
- DPBS no calcium no magnesiumGibco - Thermo FischerCatalog #14190169
- PEI MAX® - Transfection Grade Linear Polyethylenimine Hydrochloride (MW 40000)PolysciencesCatalog #24765-1 .
- Bovine Serum Albumin Fraction VSigma – AldrichCatalog #10735094001
- Sodium azideSigma AldrichCatalog #S2002
- Poly-L-lysineSigma – AldrichCatalog #P4832
Equipment
- Incubator with FPI-sensor system and display controller MB1 (BINDER GmbH. Model: CB150. Power Output: 1.40kW, 230V, 6.1 Amp)
- Leica TCS SP8 MP Multiphoton Microscope.
- See-saw rocker (VWR SSL4, or equivalent).
Consumables:
- Nunc™ Cell-Culture Treated Multidishes, 6 wellThermo FisherCatalog #140675
- Cover glasses squareVWR international LtdCatalog #631-0125
- Microscope slides SuperFrost®VWR international LtdCatalog #631-0114
- PIPETTE TIPS 100- 1000 µL BLUE SUITABLE FOR EPPENDORF STERILE 60 PIECES PER RACKgreiner bio-oneCatalog #686271
- PIPETTE TIP 10 - 100 µL SUITABLE FOR EPPENDORF 96 PIECES / ST RACKgreiner bio-oneCatalog #685261
Seeding cells for immunofluorescence microscopy
Seeding cells for immunofluorescence microscopy
Coat coverslips (sterilised in 100% ethanol prior to use) with poly-L-lysine by immersing the coverslips in poly-L-lysine solution for 01:00:00 .
Note
Note: This is only necessary when using HEK293 cells.
1h
Rinse the coated coverslips in media and place in a 6-well plate(one coverslip in each well).
Seed cells to 50-60% confluency in Growth media on coated coverslips from step 2.
Incubate Overnight .
Note
Note: For cells stably expressing GolgiTAG, skip to Step 4.
Transient transfection of cells for immunofluorescence microscopy
Transient transfection of cells for immunofluorescence microscopy
For each plasmid, prepare a transfection mix in a sterile 1.5 mL Eppendorf tube containing:
- 2 µg plasmid (see Table 1 for list of plasmids).
- 6 µL 1 mg/mL PEI Max 40K (dissolved in distilled water and filter sterile).
- 300 µL OptiMem.
Incubate the transfection mix at Room temperature for 00:30:00 .
30m
Add the transfection mix dropwise to the cells from step 3 using a P1000 sterile pipette.
Incubate cells at 37 °C for 24:00:00 .
1d
Preparing cells for Immunofluorescence imaging
Preparing cells for Immunofluorescence imaging
4h 10m
4h 10m
Remove media completely using an aspirator and wash cells 3 times with 3 mL PBS added wit 0.2% (w/v) BSA and 0.02% (w/v) sodium azide (00:05:00 per wash on a see-saw rocker).
5m
Wash cells with 3 mL PBS added wit 0.2% (w/v) BSA and 0.02% (w/v) sodium azide for 00:05:00 (1/3).
5m
Wash cells with 3 mL PBS added wit 0.2% (w/v) BSA and 0.02% (w/v) sodium azide for 00:05:00 (2/3).
5m
Wash cells with 3 mL PBS added wit 0.2% (w/v) BSA and 0.02% (w/v) sodium azide for 00:05:00 (3/3).
5m
Fix cells by adding 4% (w/v) PFA in PBS.
Incubate at Room temperature for 00:10:00 .
10m
Permeabilise cells with 1% (v/v) NP-40 in PBS + 0.2% (w/v) BSA + 0.02% (w/v) sodium azide.
Block with 1% (w/v) BSA in PBS at Room temperature for 00:30:00 .
30m
Prepare the primary antibody dilutions in 0.2% BSA (w/v) in PBS + 0.02% (w/v) sodium azide (See Table 2 for a list of antibodies and their working dilution).
Note
Note: Primary antibodies raised in different species are combined for co-staining, as follows:
- Rat anti-HA and Rabbit anti-GM130
- Rat anti-HA and Rabbit anti-GOLGIN97
- Rat anti-HA and Mouse anti-ACBD3
- Rat anti-HA and Rabbit anti-GCC185
Incubate cells at Room temperature with diluted primary antibodies for 01:00:00 .
Note
Note: This should be done in a humid chamber to avoid samples drying out. Cover a glass plate with parafilm and add 20 µL of primary antibody dilution to the relevant labelled area on the parafilm. Using tweezers, place each coverslip on the primary antibody solution (facing downward, so the cells are in contact with the antibody).
1h
Wash the coverslips 3 times with 0.2% (w/v) BSA in PBS + 0.02% sodium azide. (00:05:00 per wash).
5m
Wash the coverslips with 0.2% (w/v) BSA in PBS + 0.02% sodium azide for 00:05:00 (1/3).
5m
Wash the coverslips with 0.2% (w/v) BSA in PBS + 0.02% sodium azide for 00:05:00 (2/3).
5m
Wash the coverslips with 0.2% (w/v) BSA in PBS + 0.02% sodium azide for 00:05:00 (3/3).
5m
Prepare the secondary antibody dilutions in 0.2% BSA (w/v) in PBS + 0.02% (w/v) sodium azide (See Table 3 for a list of antibodies and their working dilution).
Incubate cells with diluted secondary antibodies (20 µL per coverslip) at Room temperature in the dark for 01:00:00 .
Note
Note: As for the incubation with primary antibodies, this should be done in a humid chamber (see step 3.7).
1h
Wash cells 3 times in 0.2% (w/v) BSA in PBS + 0.02% (w/v) sodium azide. (00:05:00 per wash).
5m
Wash cells in 0.2% (w/v) BSA in PBS + 0.02% (w/v) sodium azide for 00:05:00 (1/3).
5m
Wash cells in 0.2% (w/v) BSA in PBS + 0.02% (w/v) sodium azide for 00:05:00 (2/3).
5m
Wash cells in 0.2% (w/v) BSA in PBS + 0.02% (w/v) sodium azide for 00:05:00 (3/3).
5m
Rinse cells by dipping briefly in MilliQ water and gently dry on Kleenex wipes.
Add a drop of Gold anti-glare oil (with DAPI for nucleus staining) (Invitrogen #2086915) on a microscope glass slide labelled with the sample ID.
Mount cover slips from step 20 on the glass slide, ensuring that the side containing the cells is facing inward, making contact with the oil. Allow to dry for 00:30:00 in the dark.
30m
Slides can be stored at 4 °C or viewed immediately on a confocal microscope.
Figure 1: Immunofluorescence images of cells expressing various HA-tagged proteins. Cells were prepared for imaging using the methods described above. (A) HEK293 cells stably expressing GolgiTAG were co-immunostained for HA and Golgi markers such as ACBD3, GM130 and GOLGIN97. (B) HeLa cells transiently expressing HA-tagged proteins were co-immunostained for HA and a Golgi marker, GCC185. Scale bar is 2 µm.