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Duolink detection reagent kit

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The Duolink detection reagent kit is a laboratory tool used to detect and visualize protein-protein interactions within cells. The kit contains essential reagents and components necessary for the Duolink assay, which is a proximity ligation-based technique. The core function of the Duolink detection reagent kit is to enable the in situ detection and analysis of protein complexes.

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Lab products found in correlation

Duolink pla rabbit minus and pla mouse plus proximity probes, by Olink (3 mentions) Mouse anti flag, by Takara Bio (2 mentions) Triton x 100, by Merck Group (2 mentions) Paraformaldehyde (pfa), by Electron Microscopy Sciences (2 mentions) Prism software, by GraphPad (2 mentions) Fluoview fv1200 confocal microscope, by Olympus (2 mentions) Ab124824, by Abcam (1 mentions) Blocking solution, by Merck Group (1 mentions) Lsm 510 laser scanning microscope, by Zeiss (1 mentions) Bovine serum albumin (bsa), by Merck Group (1 mentions) Duolink in situ mounting medium with dapi, by Olink (1 mentions) All in one fluorescence microscope, by Keyence (1 mentions) Prolong gold antifade with dapi, by Thermo Fisher Scientific (1 mentions) Anti β catenin, by Abcam (1 mentions) Anti tat, by Santa Cruz Biotechnology (1 mentions) 4 6 diamidino 2 phenylindole (dapi), by Merck Group (1 mentions)

Close spelling variants of this product

Duolink Detection kit Duolink reagents kit Duolink reagent kit Duolink kit

6 protocols using duolink detection reagent kit

1

Proximity Ligation Assay for Protein-Protein Interactions

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HeLa cells transfected with indicated plasmids were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) for 15 min and permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) for 10 min at room temperature. Cells were then blocked with 3% BSA in PBS for 1 hr and incubated with appropriate combinations of mouse anti-FLAG (1:100; Clontech Laboratories) and rabbit anti-HA (1:3,000; CST) antibodies diluted in blocking solution for 1 hr at room temperature. This was followed by proximity ligation assay using the Du-olink detection reagent kit (Olink Bioscience) according to the manufacturer’s protocol. Fluorescence images were acquired using an Olympus Fluoview FV1200 confocal microscope with a 60×/1.35 UPlanSApo objective. Images were prepared and analyzed with Fiji software (http://fiji.sc). For each experiment, signals from at least 50 cells were quantified. All data were analyzed using GraphPad PRISM software.
Pagac M., Cooper D.E., Qi Y., Lukmantara I.E., Mak H.Y., Wu Z., Tian Y., Liu Z., Lei M., Du X., Ferguson C., Kotevski D., Sadowski P., Chen W., Boroda S., Harris T.E., Liu G., Parton R.G., Huang X., Coleman R.A, & Yang H. (2016). SEIPIN Regulates Lipid Droplet Expansion and Adipocyte Development by Modulating the Activity of Glycerol-3-phosphate Acyltransferase. Cell reports, 17(6), 1546-1559.
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2

CXCR4-HIF-1α Interaction Visualization

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ACHN cells were seeded on tissue culture-treated chamber slides (REF 354108; BD Falcon, BD Biosciences, BD AB, Stockholm, Sweden). The following day, the cells (1×106) were serum-starved for 12 h prior to 24-h treatment with CXCL12α (200 ng/ml). The cells were then fixed as described previously [16 (link)], and the slides were blocked in blocking solution (Sigma) for 30 min at 37 °C. After washing, the slides were incubated with Duolink PLA Rabbit MINUS and PLA Mouse PLUS proximity probes (Olink Bioscience, Uppsala, Sweden), and proximity ligation was performed using the Duolink detection reagent kit (Olink Bioscience) according to the manufacturer’s protocol. Fluorescence was detected using an LSM-510 laser scanning microscope (Carl Zeiss, Thornwood, NY). The antibodies used for the PLA were rabbit anti-CXCR4 (ab124824; Abcam) and mouse anti-HIF-1α (ab1, Abcam) antibodies.
Bao Y., Wang Z., Liu B., Lu X., Xiong Y., Shi J., Li P., Chen J., Zhang Z., Chen M., Wang L, & Wu Z. (2018). A feed-forward loop between nuclear translocation of CXCR4 and HIF-1α promotes renal cell carcinoma metastasis. Oncogene, 38(6), 881-895.
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3

Proximity Ligation Assay for Protein-Protein Interactions

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HeLa cells transfected with indicated plasmids were fixed with 4% paraformaldehyde (Electron Microscopy Sciences) for 15 min and permeabilized with 0.2% Triton X-100 (Sigma-Aldrich) for 10 min at room temperature. Cells were then blocked with 3% BSA in PBS for 1 hr and incubated with appropriate combinations of mouse anti-FLAG (1:100; Clontech Laboratories) and rabbit anti-HA (1:3,000; CST) antibodies diluted in blocking solution for 1 hr at room temperature. This was followed by proximity ligation assay using the Du-olink detection reagent kit (Olink Bioscience) according to the manufacturer’s protocol. Fluorescence images were acquired using an Olympus Fluoview FV1200 confocal microscope with a 60×/1.35 UPlanSApo objective. Images were prepared and analyzed with Fiji software (http://fiji.sc). For each experiment, signals from at least 50 cells were quantified. All data were analyzed using GraphPad PRISM software.
Pagac M., Cooper D.E., Qi Y., Lukmantara I.E., Mak H.Y., Wu Z., Tian Y., Liu Z., Lei M., Du X., Ferguson C., Kotevski D., Sadowski P., Chen W., Boroda S., Harris T.E., Liu G., Parton R.G., Huang X., Coleman R.A, & Yang H. (2016). SEIPIN Regulates Lipid Droplet Expansion and Adipocyte Development by Modulating the Activity of Glycerol-3-phosphate Acyltransferase. Cell reports, 17(6), 1546-1559.
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4

Proximity Ligation Assay for STMN1-p27 Interaction

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HuCCT1 cells were seeded and incubated on Chamber Slides (Lab-Tek II, Thermo Scientific, Waltham, MA, USA) for 24 h. The cells were fixed with 4% paraformaldehyde for 30 min and 100% methanol for 10 min. The slides were then blocked in 4% bovine serum albumin (Millipore, Biillerica, MA, USA) for 30 min and incubated for 48 h at 4°C with the appropriate combinations of mouse, rabbit and goat antibodies diluted 1:100 (STMN1 rabbit antibody; Cell Signaling Technology, Danvers, MA, USA; and p27 mouse antibody; Santa Cruz Biotechnology) in antibody dilution solution (Olink Bioscience, Uppsala, Sweden). After washing, the slides were incubated with Duolink PLA Rabbit MINUS and PLA Mouse PLUS proximity probes (Olink Bioscience) and a proximity ligation was performed using the Duolink Detection Reagent Kit (Olink Bioscience) according to the manufacturer's protocol. Nuclei were stained with Duolink In Situ Mounting Medium with DAPI (Olink Bioscience). Images were acquired with an All-in-one Fluorescence Microscope (Keyence Corporation, Osaka, Japan).
Watanabe A., Suzuki H., Yokobori T., Tsukagoshi M., Altan B., Kubo N., Suzuki S., Araki K., Wada S., Kashiwabara K., Hosouchi Y, & Kuwano H. (2014). Stathmin1 regulates p27 expression, proliferation and drug resistance, resulting in poor clinical prognosis in cholangiocarcinoma. Cancer Science, 105(6), 690-696.
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5

Visualizing Protein Interactions in Skin

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Frozen skin sections were prepared as described above and protein interactions visualized by immunofluorescence using Duolink in situ PLA technology (Olink Bioscience, Uppsala, Sweden). Sections were blocked for 2 hours in 3% BSA/PBS then incubated with antibodies to LH3 (rabbit polyclonal, 1:10 dilution) and type VII collagen (mouse LH7.2, 1:800 dilution) overnight at 4°C. After washing, the slides were incubated with Duolink PLA Rabbit MINUS and PLA Mouse PLUS proximity probes (Olink Bioscience) at a 1:5 dilution at 37°C for 2 hours. Proximity ligation was then performed using the Duolink detection reagent kit (Olink Bioscience) according to manufacturer’s instructions except for the amplification step which was performed for 2 hours at 37°C. Slides were mounted using ProLong Gold Antifade with DAPI (Life Technologies, Paisley, UK) and imaged on the Zeiss Axioskop 2 fluorescent microscope using AxioVision Rel. 4.7 software (Carl Zeiss, GmbH, Jena, Germany).
Watt S.A., Dayal J.H., Wright S., Riddle M., Pourreyron C., McMillan J.R., Kimble R.M., Prisco M., Gartner U., Warbrick E., McLean W.H., Leigh I.M., McGrath J.A., Salas-Alanis J.C., Tolar J, & South A.P. (2015). Lysyl Hydroxylase 3 Localizes to Epidermal Basement Membrane and Is Reduced in Patients with Recessive Dystrophic Epidermolysis Bullosa. PLoS ONE, 10(9), e0137639.
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6

Visualizing Protein-Protein Interactions in Cancer Cells

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MCF-7 and MDA-MB-231 cells were cultured in 35-mm plates with cover slides on the plate bottom and treated with NLS-BLBD-6, TAT-BLBD-6 and TAT-NLS-BLBD-6 peptide for 24 hr. Subsequently, cells were fixed with 4% paraformaldehyde and permeabilized with 0.2% Triton X-100 for 20 min at room temperature. For the immunofluorescence assay, the slides were incubated with primary antibodies, anti-β-catenin (Epitomics) and anti-TAT (Santa Cruz), for 6 hr, and Alexa-488 fluorescence secondary antibodies for 1 hr. DAPI (Sigma, St Louis, MO) was used to stain the nucleus for 1 min at room temperature. For the TUNEL assay, apoptosis was analyzed by the Apo-BrdU-red DNA Fragmentation Assay kit, according to the manufacturer’s protocol (BioVision, Mountain View, CA, USA). For the PLA, the protein–protein interaction assay was analyzed by Duolink® using PLA® Technology, according to the manufacturer’s protocol (Olink Bioscience, Uppsala, Sweden). Briefly, the slides were incubated with primary antibodies, anti-β-catenin (Epitomics) and anti-TAT (Santa Cruz), and secondary antibodies, Duolink PLA Rabbit MINUS and PLA Mouse PLUS proximity probes. Finally, the proximity ligation was performed by the Duolink detection reagent kit (Olink Bioscience). The immunofluorescence image and TUNEL staining was photographed by a microscope (IX-71, Olympus, Tokyo, Japan).
Hsieh T.H., Hsu C.Y., Tsai C.F., Chiu C.C., Liang S.S., Wang T.N., Kuo P.L., Long C.Y, & Tsai E.M. (2016). A novel cell-penetrating peptide suppresses breast tumorigenesis by inhibiting β-catenin/LEF-1 signaling. Scientific Reports, 6, 19156.
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