Proximity ligation assay (PLA) to detect ASRGL1 and TDP-43 interaction was performed with the Duolink Proximity Ligation Assay kit (Millipore Sigma) in HEK293 transfected cells and brain slides, following manufacturer’s instructions. Briefly, samples were blocked with Duolink® Blocking Solution for 60 min at 37 °C. Then, primary antibodies were applied (ASRGL1: Atlas HPA05572 (rabbit). TDP-43: Proteintech (mouse)) at 1:200 in Duolink antibody diluent, and samples were incubated overnight in a humidity chamber at room temperature. Then, minus and plus probes were diluted 1:5 in the Duolink antibody diluent. The slides were washed twice in wash buffer A and incubated with the probes for 1 h at 37 °C. Samples were again washed twice in wash buffer A and incubated with ligase for 30 min at 37 °C. Samples were once again washed twice in wash buffer A. Then, polymerase was added, and slides were incubated for 100 min at 37 °C. Slides were washed twice in wash buffer B 1x and once in wash buffer B 0.01x. Slides were mounted with Duolink mounting media with DAPI and analyzed by confocal microscopy. Since the levels of ASRGL1 are higher in normal controls than in ALS individuals, negative controls were produced from slides from normal controls: (1) No primary antibodies, (2) Only TDP-43 antibody, (3) Only ASRGL1 antibody, (4) TDP-43 antibody + Control isotype IgG.
Duolink proximity ligation assay kit
Manufactured by Merck Group
Sourced in Germany, Belgium, United States
The Duolink Proximity Ligation Assay kit is a tool used to detect and visualize protein-protein interactions in cells. It utilizes a unique proximity ligation technique to amplify and detect these interactions, providing a sensitive and specific method for analysis.
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Lab products found in correlation
Lsm 880 confocal microscope, by Zeiss (4 mentions)
Pparα, by Santa Cruz Biotechnology (3 mentions)
Pparα, by Merck Group (3 mentions)
Alexa fluor 594 conjugated goat anti rabbit igm, by Thermo Fisher Scientific (3 mentions)
Alexafluor 488 conjugated goat anti mouse igg1, by Thermo Fisher Scientific (3 mentions)
Ncor1, by Cell Signaling Technology (3 mentions)
Ab14734, by Abcam (2 mentions)
Farred, by Merck Group (2 mentions)
Arthrobacter uereafaciens sialidase, by Roche (2 mentions)
In situ pla probe anti mouse minus, by Merck Group (2 mentions)
Anti goat plus, by Merck Group (2 mentions)
Tdp 43, by Proteintech (1 mentions)
Ab20346, by Abcam (1 mentions)
Mitotracker, by Beyotime (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Wuhan Servicebio Technology (1 mentions)
A1r confocal microscope, by Nikon (1 mentions)
Alexa flour 488 conjugated phalloidin, by Thermo Fisher Scientific (1 mentions)
Sp5 2 confocal microscope, by Leica (1 mentions)
Cas block, by Thermo Fisher Scientific (1 mentions)
Duo92002, by Merck Group (1 mentions)
26 protocols using duolink proximity ligation assay kit
1
Proximity Ligation Assay for ASRGL1-TDP-43 Interaction
2
Proximity Ligation Assay for Protein Interactions
3
Proximity Ligation Assay for Protein Interactions
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Protein interactions were detected via Duolink proximity ligation assay kit (Merck, DUO92105) using an anti-rabbit plus probe and an anti-goat minus probe as according to the manufacturer’s instructions. Briefly, cells isolated by dissociation were fixed for immunocytochemistry as above, then blocked with the kit blocking solution for 1 h at room temperature. Antibodies were applied at a ratio of 1:100 with kit antibody diluent solution and incubated overnight at 4°C. Following this, Duolink probes were added to cells at a ratio of 1:5 with kit diluent solution and incubated for 1 h at 37°C in a humidified chamber and excess was washed in kit wash buffer. Cells were incubated in a humidified chamber heated to 37°C in kit solutions for ligation of probes (30 min), and amplification of the signal (100 min), then cells were counterstained with 4′-6-Diamidino-2-phenylindole (DAPI). The plus and minus probes were bound to the anti-WNT3A antibody, and antibody anti-FRZB, respectively. Where the distance between the two bound probes is <40 nm a red signal is generated, which indicates an interaction of proteins of interest [30 (link)].
4
Proximity Ligation Assay for USP53-CYCS Interaction
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A Duolink® proximity ligation assay kit (Merck, DUO92102, Darmstadt, Germany) was used to perform the proximity ligation assay by following the manufacturer’s instructions. Anti-USP53 Rabbit polyclonal Ab (Thermo Fisher, PA5-57424) was conjugated with a PLUS nucleotide probe, while anti-CYCS mouse monoclonal Ab was conjugated with a MINUS nucleotide probe. All images were visualized and captured using an Olympus Fluoview 1000 laser scanning confocal microscope (Leica, Wetzlar, Germany). The MitoTracker was purchased from Beyotime (C1048, Shanghai, China), while DAPI was obtained from Servicebio (G1012, Wuhan, China).
5
Proximity Ligation Assay for Pyrin and β2MG
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Proximity ligation assay was performed using Duolink® proximity ligation assay kit (Merck). Anti-pyrin mouse monoclonal Ab was conjugated with PLUS nucleotide probe, and anti-β2MG mouse monoclonal Ab was conjugated with MINUS nucleotide probe, respectively. Unstimulated and stimulated neutrophils were subjected to the proximity ligation assay according to the manufacturer’s protocols.
6
Detecting Protein Interactions with Duolink
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Proximity ligation assay was performed to detect in situ protein interaction of TM, pT567ezrin, and ROCK2 with a fluorescence Duolink proximity ligation assay kit (DUO92101, Merck) according to the manufacturer's instructions.
7
Duolink Proximity Ligation Assay Protocol
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The Duolink proximity ligation assay (PLA) kit (DUO92008, Sigma, Bornem, Belgium) was used according to the manufacturer’s instructions. Primary antibodies were used at dilution 1:75 (Myoferlin, MFN1/2) or 1:100 (others). Oligonucleotide conjugated secondary antibodies were provided by the kit allowing the detection of a red signal if less than 40 nm separate both proteins of interest. Pictures were acquired using Nikon A1R confocal microscope. In each microscopic field, proximity dots were counted using ImageJ software [24 (link)] and divided by the number of nuclei to calculate an average proximity dots number per cell. To analyze PLA, we wrote a script to automatically analyze the data in a standardized way. The script is available at [26 ].
8
Proximity Ligation Assay for Protein Interactions
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Protein interactions were tested using the DuoLink proximity ligation assay (PLA) kit (DUO92008; Sigma). Thirty-six hours after overexpression of the VP3-HA plasmid, the described immunofluorescence fraction was fixed, permeabilized, and blocked with DuoLink Closure Solution liquid for 60 min at 37°C. Cells were incubated with the corresponding primary antibody diluted in DuoLink dilution buffer. After washing, cells were incubated with species-specific PLA probes (positive and negative) for 1 h at 37°C under hybridization conditions. Then ligase was added and incubated at 37°C for 30 min to ligate the hybridized oligonucleotides. Amplification polymerase was added to produce a ligated product extending from the oligonucleotide arm of the PLA probe. Nuclei were stained with Duolink in in situ mounting medium containing DAPI.
9
Visualizing Viral Capsid-Nup358 Interactions
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Duolink proximity ligation assay (PLA) kit was purchased from Sigma and assay performed as described by the manufacturer (Olink Bioscience). In brief, cells grown on coverslips were fixed with 3.7% PFA 3h post synchronized infection. To detect interaction between viral capsid protein p24 and Nup358, cells were permeabilized and blocked in 3% BSA followed by incubation with primary antibodies targeting viral protein p24 (mouse monoclonal) and Nup358 (rabbit polyclonal). After primary staining, coverslip containing cells were washed and incubated (1h, 37°C) with secondary anti-mouse conjugated with minus and anti-rabbit conjugated with plus Duolink II PLA probes. Coverslips were washed again and incubated with ligation-ligase solution (30min, 37°C) followed by washing and subsequent incubation with amplification-polymerase solution (100min, 37°C) containing Duolink II insitu detection reagent Red. Finally, coverslips were washed and mounted with Duolink II mounting medium containing DAPI. Interactions were detected as fluorescent spots (λ excitation/emission 598/634 nm) under a fluorescence microscope.
10
Protein-Protein Interaction Mapping in Hippocampus
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Protein–protein interactions between endogenous proteins were detected by using a Duolink proximity ligation assay (PLA) kit (Sigma-Aldrich). The PLA probe anti-Rabbit MINUS binds to the TASK-3 antibody; whereas, the PLA probe anti-Goat PLUS binds to the TWIK-1 antibody. After pre-incubation with a blocking agent for 1 h, the frozen sectioned hippocampal tissues were incubated overnight with primary antibodies to TASK-3 (1:100, Abcam, Cambridge, UK) and TWIK-1 (1:100, Santa Cruz Biotechnology, Dallas, TX, USA). Duolink PLA probes detecting rabbit or goat antibodies were diluted in the antibody diluent to a concentration of 1:5 and applied to the slides, followed by incubation for 2 h in a preheated humidity chamber at 37 °C. Duolink hybridization stock (dilution 1:5) was used to hybridize the two Duolink PLA probes, which were incubated in a preheated humidity chamber for 30 min at 37 °C and then incubated in a ligation solution consisting of Duolink ligation stock (1:5) and Duolink Ligase (1:40) for 90 min at 37 °C. Detection of the amplified probe was performed with the Duolink detection kit with the Duolink detection stock diluted at 1:5 and applied for 1 h at 37 °C. The final washing steps were performed in saline sodium citrate buffer.
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