HepG2 cells were seeded on an 8-well chamber slide (154534, Thermo Fisher Scientific). After three washes with PBS, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature. Then, the cells were incubated with 1 × Permeabilization Buffer (00-8333-56, Thermo Fisher Scientific) for 15 min, followed by blocked with Duolink block solution for 1 h at room temperature and incubated overnight at 4 °C with the following antibodies: (1) mouse anti-Flag (F3165, Sigma-Aldrich) and rabbit anti-eIF3a (ab86146, Abcam); (2) rabbit anti-METTL16 (HPA020352, Sigma-Aldrich) and mouse anti-eIF3b (sc-137214, Santa Cruz Biotechnology). The next day, cells were washed twice with a large volume of PBS and incubated in PLA probes (DUO92002 and DUO92004, Sigma-Aldrich) for 1 h at 37 °C. Then, the cells were washed with 1 × Duolink for two times In Situ Wash Buffer A (DUO82049, Sigma-Aldrich) and incubated with ligation mix at 37 °C for 30 min. Subsequently, the cells were washed with 1 × Duolink In situ Wash Buffer A twice and incubated with amplification mix (DUO92008, Sigma-Aldrich) at 37 °C for 100 min. Finally, the cells were washed twice with 1 × Duolink in situ wash buffer B, washed once with 0.01 × Buffer B and mounted with Duolink in situ mounting medium with DAPI (DUO82040, Sigma-Aldrich). The pictures were captured under LSM 880 confocal microscope (Zeiss, Germany).
Duo82049
Manufactured by Merck Group
The DUO82049 is a laboratory equipment product manufactured by the Merck Group. It is a scientific instrument designed for specific laboratory applications. The core function of this product is to perform precise measurements or analyses as required by researchers and scientists in various fields. No further details are provided to maintain an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Duo82040, by Merck Group (2 mentions)
Lsm 880 confocal microscope, by Zeiss (2 mentions)
Ab86146, by Abcam (1 mentions)
Duo92002, by Merck Group (1 mentions)
Duo92004, by Merck Group (1 mentions)
Duo92008, by Merck Group (1 mentions)
Hpa020352, by Merck Group (1 mentions)
Sc 137214, by Santa Cruz Biotechnology (1 mentions)
F3165, by Merck Group (1 mentions)
Hif 1α, by GeneTex (1 mentions)
Cas block, by Thermo Fisher Scientific (1 mentions)
Fv3000, by Olympus (1 mentions)
Mab2605, by GeneTex (1 mentions)
Fv1000, by Olympus (1 mentions)
5 protocols using duo82049
1
Proximity Ligation Assay for Protein Interactions
2
Visualizing Protein-Protein Interactions in Hypoxia
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PLA was performed to visualize protein-protein interaction in MDCK cells. In brief, cells were seeded in 3-cm glass-bottom dishes and treated with or without hypoxia-mimetic agent CoCl2 for 8 h. After treatment, cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.5% Triton X-100 for 10 min at room temperature. After blocking with CAS-Block (#00-8120; Invitrogen) for 1 h, cells were incubated with primary antibodies (HIF-1α [#GTX127309; GeneTex] and YAP [#H00010413; Abnova]) at 4 °C overnight. On the next day, dishes were washed twice with washing buffer (#DUO82049, Sigma-Aldrich), then incubated with PLA probes (1:5 in antibody diluent) for 1 h at 37 °C. After PLA probes incubation, dishes were then incubated with ligation solution for 30 min at 37 °C, followed by the amplification solution for 100 min at 37 °C. Hoechst 33342 were then added to the dishes for 15 min. The images were acquired using a confocal microscope (FV3000; Olympus).
3
Visualizing Protein-Protein Interactions in Cancer Cells
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In situ PLA was performed to visualize protein-protein interactions in MDA-MB-231 and A549 cells. Briefly, cells were seeded onto 3-cm dishes overnight for adherence with ~80% confluence. Cells were then washed with PBS twice, and fixed with 4% paraformaldehyde for 15 min at room temperature. The fixed cells were permeabilized with PBS containing 1% Triton X-100 (Calbiochem, 9410-OP) for 30 min, subsequently blocked in Blocking Solution (Sigma-Aldrich, DUO82007) at 37°C for 1 h and incubated with primary antibodies (anti-BIRC5 [Cell Signaling Technology, 2808]; anti-ATG5 [Millipore, MAB2605; Gene Tex, GTX113309]; anti-ATG12 [Gene Tex, GTX629815], and anti-ATG16L1 [Millipore, ABC25]) overnight at 4°C. On the following day, cells were washed twice with washing buffer (Sigma-Aldrich, DUO82049), and incubated with PLA probes in a ratio of 1:5 in antibody diluent for 1 h at 37°C. The cells were then incubated with ligation solution at 37°C for 30 min and subsequently with amplification solution at 37°C for 100 min. Duolink in situ mounting medium (Sigma-Aldrich, DUO82040) together with DAPI were added to the cells at room temperature for 10 min. Cell images were captured with a confocal microscope (FV1000, Olympus).
4
Proximity Ligation Assay for Protein Interactions
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Cells grown on coverslips were fixed with 4% paraformaldehyde in PBS at room temperature for 10 min, followed by PBS washing for 4 × 5 min. Cells were then permeabilized with permeabilization buffer at 4 °C for 6 min, followed by PBS washing for 4 × 5 min. PLA was performed with Duolink reagents (Sigma Aldrich) according to the manufacturer’s protocol. Briefly, coverslips were blocked with Duolink® blocking solution at 37 °C for 60 min and incubated with primary antibodies in Duolink antibody diluent at 4 °C overnight, followed by wash buffer A (Sigma, Cat# DUO82049) washing for 2 ×5 min at room temperature. Ligation was performed with ligase in ligation buffer at 37 °C for 60 min, followed by wash buffer A washing for 2 ×5 min at room temperature. Amplification was performed with polymerase in amplification buffer at 37 °C for 100 min, followed by wash buffer B washing for 2 ×10 min at room temperature. After washing with 0.01x Wash Buffer B for 1 min, coverslips were mounted using ProLong™ Gold Antifade Mountant with DAPI. Images were collected by Nikon A1-Confocal using ×60 objective and analyzed by NIH Elements AR software (AR 5.21.03 64 bit).
5
Proximity Ligation Assay for ER-Mito
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ER-Mito cells were plated, fixed, quenched and permeabilized using the immunofluorescence protocol. PLA was conducted using Duolink In Situ PLA Probe kits, Duolink In Situ detection reagents far red (Sigma, DUO92013-100RXN) and Duolink in Situ Wash buffer (Sigma, DUO82049) according to the manufacturer’s instructions. Blocking was conducted using 1x blocking buffer for 1 h at 37 °C. Primary antibodies were added and incubated at 4 °C in antibody dilutant overnight in a humidified chamber. The next day, the cells were washed with Duolink Buffer A (2 × 5 min washes) and incubated with Duolink anti-mouse plus and anti-rabbit minus probes for 1 h at 37 °C. The coverslips were further washed in buffer A (2 × 5 min) and incubated with 1x ligase for 30 min at 37 °C. After two 5 min washes, the coverslips were incubated with 1x polymerase for 100 min at 37 °C. After this last incubation, the coverslips were washed in Duolink buffer B (2 × 10 min), in 0.01x buffer B for 1 min, then in ddH2O and incubated with Hoechst if needed. Coverslips were mounted using Dako Mounting media (S3023).
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