PLA was conducted using Duolink PLA technology with Orange mouse/rabbit probes (Sigma-Aldrich, DUO92102) according to the manufacturer’s instructions. Images were captured using SP8 6000 confocal imaging with 0.4um Z-stacks. Maximum projects were made for each image (100–200 cells) and quantified using FIJI by ImageJ [51 (link)] as described previously [52 (link)]. Quantification was conducted on a minimum of 50 cells. Data is represented as number of interactions (dots) per cell.
Duo92102
Manufactured by Merck Group
The DUO92102 is a piece of laboratory equipment manufactured by Merck Group. It is designed to perform specific functions within a laboratory setting. The core function of this product is to facilitate various laboratory tasks, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
β catenin, by Cell Signaling Technology (1 mentions)
Csm710, by Zeiss (1 mentions)
D3571, by Thermo Fisher Scientific (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
Lsm900 microscope, by Zeiss (1 mentions)
Anti nlrp3 antibody, by Adipogen (1 mentions)
Plan apochromat 20 0.8 na objective, by Zeiss (1 mentions)
Lsm 880 confocal microscope, by Zeiss (1 mentions)
Prolong gold antifade reagent, by Thermo Fisher Scientific (1 mentions)
Lsm 710, by Zeiss (1 mentions)
8 protocols using duo92102
1
Quantifying Protein-Protein Interactions with Duolink PLA
2
Trio-Myh9 Interaction Validation Protocol
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The PLA was performed to confirm the interaction of Trio with Myh9 according to the manufacturer's protocol (Sigma Aldrich #DUO92102). In short, NCCs were seeded onto slides in a 24-well plate at a density of 2 × 104 cells/well, washed 2 times with PBS and fixed in 4% PFA for 20-30 min and then permeabilized in 0.5% Triton X-100 for 20 min, washed 2 times with PBS, blocked with blocking reagents for 60 min at 37 ºC. After blocking, cells were incubated with primary antibodies against Trio (1:100, Abcam) and Myh9 (1:100, proteintech) or RAC1 (1:100, Cytoskeleton) and β-catenin (1:100, Cell Signaling Technology) overnight at 4 ºC, followed by corresponding secondary antibodies conjugated with PLA probes for 60 min at 37 ºC. Cells were then incubated with ligation reagent for 30 min and with amplification reagent for 100 min at 37 ºC in dark. Next, cells were washed three times with wash buffer and incubated with a mounting medium with DAPI for 15 min in dark. Finally, Duolink and DAPI signals were detected using confocal microscopy (ZEISS CSM710).
3
Proximity Ligation Assay for β1 Integrin-Tensin Interaction
4
Probing SMOC-2 and Wnt Receptor Interactions
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Duolink PLA assay was performed to detect the interactions between SMOC-2 and LRP6/Fzd6 in stem cells and non-stem cells (DUO92102, Sigma). After fixation and permeabilization, cells were incubated with DuoLink blocking buffer for 60 min at 37 °C, followed by incubation with primary antibodies (SMOC-2, 1:200, Abcam, ab78069; LRP6, 1:200, Abcam, ab134146; Fzd6 1:100, Bioss, bs-13218R) for overnight at 4 °C. Cells were incubated with Duolink PLA Probe (anti-rabbit PLA probe PLUS and anti-mouse PLA probe MINUS)) for 1 h at 37 °C in a pre-heated humidity chamber, followed by incubation with Duolink Ligation buffer and Amplification buffer according to manufacturer's protocol. Finally, mounted the slides with Duolink In Situ Mounting Medium with DAPI, and analyzed in a fluorescence microscope.
5
Protein-Protein Interaction in THP-1 Cells
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PMA-stimulated THP-1 cells were cultured on an eight-well chamber slide. In the presence or absence of LPS + ATP, the cells were subjected to PLA according to the manufacturer’s instructions (DUO92102, Sigma‒Aldrich). Briefly, cells were fixed with 4% PFA, incubated with Duolink blocking solution for 1 h at 37 °C in an cell culture chamber, and then incubated again with primary antibodies, including the anti-NCOA6 antibody (1:100, Bethyl Laboratories) and anti-NLRP3 antibody (1:100, Adipogen), overnight at 4 °C. After washing with PBS twice for 5 min at room temperature, the cells were treated with PLA probes for 1 h at 37 °C. The cells were washed with PBS twice for 5 min at room temperature, and ligase was added to the cells for 30 min at 37 °C. After washing twice with PBS, the cells were incubated with polymerase for 100 min at 37 °C. Images were taken with a Zeiss LSM 900 microscope and analyzed with ImageJ software. The number of red fluorescent dots for each Z-stack was divided by the total number of cells labeled with DAPI per image.
6
Proximity Ligation Assay in HeLa Cells
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HeLa cells were fixed in 3.7% formaldehyde for 10 min at room temperature and permeabilized by 0.3% Triton X-100 solution in PBS. After washing three times with PBS, a standard PLA procedure was performed as recommended by the manufacturer (DUO92102; Sigma–Aldrich). In brief, cells were treated with blocking solution for 1 h. One of the primary antibodies (anti-YB1, anti–Ago-1, anti–Ago-2, Smad-1, or Lamina-1) was diluted 1:200 and added to the cells for an overnight incubation at 4 °C. Diluted (1:5) PLA probes were added to the cells after two consecutive 5-min washes (wash buffer A) and incubated for 1 h at 37 °C. Following an additional 5-min wash with buffer A, diluted (1:5) ligation stock was added to the samples and the incubation was extended for an additional 30 min. Cells were washed twice for 2 min with buffer A. The amplification step was performed by applying a mix of 1:5 diluted amplification stock and 1:80 diluted polymerase to the cells and incubating for 100 min at 37 °C. Cells were washed with buffer B several times and mounted for imaging using Duolink In Situ Mounting Medium with DAPI. After ∼15 min, Z-section fluorescent images were acquired with a Zeiss LSM880 confocal microscope using a Plan-Apochromat 20×/0.8 N.A. objective.
7
Proximity Ligation Assay for Protein Interactions
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For proximity ligation assays (PLAs, Duolink, Sigma, DUO92102), N2a cells were treated as for immunostaining until day 2, after which the manufacturer's instructions were followed. Each experiment included a negative control (2 proteins known not to interact, e.g. Parkin and histone H3) and a positive control (known binding partners Parkin and alpha‐tubulin). PLAs were performed on fixed, free‐floating pR5 and wild‐type (WT) littermate brain sections. WT sections served as the negative control and were tested for Parkin and α‐tubulin as the positive control.
8
Proximity Ligation Assay for E2f4 and Deup1
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PLA was performed according to the manufacturer’s protocol (Sigma,
DUO92102)46 (link). Briefly, ALI day 4 cultures from
E2f4f/f;
R26CreERT2/+ with or without Tamoxifen
treatment were fixed with 4% PFA room temperature for 10 min.
Samples were incubated with primary antibodies (anti E2f4, anti Deup1 or the
relevant control IgGs, as indicated) using the immunofluorescence microscopy
protocol described above, followed by incubation with the PLA probe set
(anti-rabbit plus strand/anti-mouse minus strand) at 37 °C for
2 h. Ligation of probes in close proximity was carried out at
37 °C for 30 min, followed by a 100-min amplification step.
Subsequently, we performed immunofluorescence staining for anti-Pcm1 antibody as
described above. All processed slides were mounted using ProLong Gold antifade
reagent (Life Technologies, catalogue no. P36930), and images were captured
using a confocal microscope system (Carl Zeiss, LSM 710).
DUO92102)46 (link). Briefly, ALI day 4 cultures from
E2f4f/f;
R26CreERT2/+ with or without Tamoxifen
treatment were fixed with 4% PFA room temperature for 10 min.
Samples were incubated with primary antibodies (anti E2f4, anti Deup1 or the
relevant control IgGs, as indicated) using the immunofluorescence microscopy
protocol described above, followed by incubation with the PLA probe set
(anti-rabbit plus strand/anti-mouse minus strand) at 37 °C for
2 h. Ligation of probes in close proximity was carried out at
37 °C for 30 min, followed by a 100-min amplification step.
Subsequently, we performed immunofluorescence staining for anti-Pcm1 antibody as
described above. All processed slides were mounted using ProLong Gold antifade
reagent (Life Technologies, catalogue no. P36930), and images were captured
using a confocal microscope system (Carl Zeiss, LSM 710).
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