HUVEC were fixed with 4% PFA for 15 min at room temperature. Fixed cells were blocked with Duolink Blocking Buffer (Sigma, Duo92002) for 1 h at room temperature and incubated with rabbit anti-MLKL and mouse anti-RBM6 antibodies overnight. Duolink In Situ proximity ligation assay (PLA) probe anti-rabbit PLUS IgG (Sigma, Duo92002) and anti-mouse MINUS IgG (Sigma, Duo92004) were added to the cells, and cells were incubated for 1 h at 37 °C. Duolink In Situ Detection Reagent Red (Sigma, Duo92008) was used for the detection of PLA signal. Ligation reagents containing ligase were added, and cells were incubated for 30 min at 37 °C. Afterwards, amplification reagents containing polymerase were added, and cells were incubated for 90 min at 37 °C. The PLA signal was observed as red pots by Leica SP8 confocal microscope and was analyzed using Image J.
Duolink in situ proximity ligation
Manufactured by Merck Group
Duolink in situ proximity ligation is a laboratory technique that enables the detection and visualization of protein-protein interactions within cells. It combines immunodetection with a DNA-based amplification system to generate a signal when two target proteins are in close proximity.
Automatically generated - may contain errors
Lab products found in correlation
Nunc lab tec 2 16 well glass chamber slides, by Thermo Fisher Scientific (3 mentions)
Duolink blocking buffer, by Merck Group (1 mentions)
Duolink in situ detection reagent red, by Merck Group (1 mentions)
Sp8 confocal microscope, by Leica camera (1 mentions)
Rabbit anti amot, by Fortis Life Sciences (1 mentions)
Mouse anti smad1, by Santa Cruz Biotechnology (1 mentions)
Mouse anti yap taz, by Santa Cruz Biotechnology (1 mentions)
Axiovert 200m microscope, by Zeiss (1 mentions)
Smad1, by Abcam (1 mentions)
Bmprii, by BD (1 mentions)
Smad4, by Cell Signaling Technology (1 mentions)
5 protocols using duolink in situ proximity ligation
1
Proximity Ligation Assay for MLKL-RBM6 Interaction
2
Quantifying Growth Factor-Induced Protein Interactions
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HUVECs were seeded at 200,000 cells/cm2 in HUVEC growth medium in Nunc Lab-Tec II 16-well glass chamber slides (Thermo Scientific). Cells were serum-starved for 2 h in HUVEC starvation medium, followed by stimulation with the indicated growth factors for 60 min. Subsequently, Duolink in situ proximity ligation (Sigma-Aldrich) was performed as previously described (Thymiakou and Episkopou, 2011 (link)). The number of heteromers was quantified using BlobFinder image analysis software as previously described (Allalou and Wählby, 2009 (link)). At least 15-20 images per experimental condition were quantified and normalized to untreated control cells.
3
Proximity Ligation Assay for SMAD1-YAP/TAZ-AMOT Heteromers
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MCF7 cells were seeded in Nunc Lab-Tec II 16-well glass chamber slides (Thermo Fisher Scientific) and incubated for 48 h before serum starvation followed by BMP stimulation for the indicated time points. Cells were then fixed using 4% paraformaldehyde and permeabilized with 0.5% Triton X-100. Subsequently, PLA was performed using Duolink in situ proximity ligation (Sigma-Aldrich), as previously described (Thymiakou and Episkopou, 2011 (link)), using mouse anti-SMAD1 (sc7965; Santa Cruz Biotechnology), mouse anti-YAP/TAZ (sc101199; Santa Cruz Biotechnology), and rabbit anti-AMOT (Bethyl Labs) at a dilution of 1:200. Imaging occurred with an inverted epifluorescence Axiovert 200M microscope (Zeiss). The number of heteromers was quantified using BlobFinder image analysis software as previously described (Allalou and Wahlby, 2009 (link)). At least 500 cells per experimental condition were quantified of each replicate and normalized to starved cells.
4
In Situ Proximity Ligation for Protein Interactions
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Immortalised human myoblasts seeded in Nunc Lab-Tec II 16-well glass chamber slides (Thermo Scientific) were subjected to Duolink in situ proximity ligation (Sigma-Aldrich) as previously described77 (link) using the following antibodies: BMPRII (#612292, BD Biosciences), IRS4 EP907Y (#TA303856, Origene), Smad1 (ab55476, Abcam), Smad4 (#9515, Cell Signaling).
5
Proximity Ligation Assay for Protein Interactions
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Cells were seeded at 200,000 cells/cm2 in M199 basal medium on glass coverslips. Monolayers of cells were serum starved for 6 h, followed by stimulation with the indicated growth factors for 15 min. Subsequently, Duolink in situ proximity ligation (Sigma Aldrich) was performed as previously described [126 ]. Specificity of antibodies was verified by single antibody controls as well as positive control, i.e., TGFβ-dependent SMAD2/3-SMAD4 translocation into the nucleus.
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