CHO-K1 cells were incubated with PT for indicated periods of time. Cells were fixed with 4% PFA, permeabilized and blocked with 10% NGS and 1% BSA in PBST. Subsequently, cells were incubated with mouse anti-PTS1 (Santa Cruz or Abcam) and rabbit anti-Cyp40 (Thermo Fisher Scientific), rabbit anti-Hsp90 (Thermo Fisher Scientific), rabbit anti-Hsp70 (Enzo Life Sciences) or rabbit anti-FKBP51 (Santa Cruz) antibodies for 1 h at 37 °C. PLA assay was performed according to the manufacturer’s protocol (Duolink using PLA technology, Sigma-Aldrich, Merck) and as described before46 (link),47 (link).
Duolink using pla technology
Manufactured by Merck Group
Duolink is a laboratory equipment product from Merck Group that utilizes Proximity Ligation Assay (PLA) technology. PLA is a technique for detecting and analyzing protein-protein interactions and modifications within cells. The core function of Duolink is to enable the detection and visualization of these molecular interactions.
Automatically generated - may contain errors
Lab products found in correlation
Mouse anti pts1, by Santa Cruz Biotechnology (2 mentions)
Rabbit anti cyp40, by Thermo Fisher Scientific (2 mentions)
Triton x 100, by Merck Group (1 mentions)
Anti myc, by Merck Group (1 mentions)
Duolink in situ mounting medium with dapi, by Merck Group (1 mentions)
Tcs sp5 confocal microscope, by Leica (1 mentions)
Anti ha, by Abcam (1 mentions)
Duolink in situ detection reagents red, by Merck Group (1 mentions)
18 well μ slides, by Ibidi (1 mentions)
Rabbit anti pt, by Abcam (1 mentions)
Mouse anti α defensin 5, by Abcam (1 mentions)
4 protocols using duolink using pla technology
1
Proximity Ligation Assay for Protein-Protein Interactions
2
Proximity Ligation Assay for Protein Interactions
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For visualization of protein–protein interaction in situ, Proximity Ligation Assay (PLA) was performed according to manufacturer's instructions (Duolink using PLA Technology, Sigma). Briefly, 48 h after transient HA-TET1 and/or myc-GADD45a and myc-NFYC (control protein) transfection, HEK293T cells were fixed in 100% MeOH (Sigma) for 10 min at −20 °C, permeabilized with 0.5% TritonX100 (Sigma) for 10 min, blocked and incubated with primary antibodies anti-HA (Abcam) and anti-myc (Sigma) (both diluted 1:1000) overnight at 4 °C. Then, cells were incubated with respective secondary antibodies (Duolink In Situ PLA Probes Anti-Mouse and Anti-Rabbit both PLUS and MINUS, SIGMA) followed by a ligation and subsequent amplification step to visualize protein interaction by red fluorescence signals (Duolink In Situ Detection Reagents Red, Sigma). To visualize individual proteins by Self-PLA, the respective primary antibody was combined with two secondary antibodies (PLA probes both PLUS and MINUS). Nuclei were counterstained by DAPI-containing mounting medium (Duolink In Situ Mounting Medium with DAPI, Sigma) and analysis was performed using a TCS SP5 confocal microscope (Leica) and 63× oil immersion objective lens. The speckles are likely limited to a fraction of cells due to incomplete transfection efficiency.
3
Protein Interaction Analysis of PT and Defensin
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Cells were seeded in 18-well μ-slides (ibidi GmbH, Gräfelfing, Germany) and incubated with PT and defensin for 4 h at 37 °C. Afterwards, cells were washed with PBS, fixed with 4% PFA, permeabilized and blocked with 10% NGS and 1% BSA in PBS-T. Then, cells were incubated with rabbit anti-PT (Abcam) and mouse anti-HNP (= α-defensin-1) (Santa Cruz) or mouse anti-α-defensin-5 (Abcam) primary antibodies for 1 h at 37 °C. PLA was performed according to the manufacturer’s protocol (Duolink using PLA technology, Sigma-Aldrich, Merck). In brief, cells were incubated with PLA secondary antibodies (anti-rabbit for detecting anti-PT and anti-mouse for detecting anti-defensin) having attached specific oligonucleotide sequences to each. If they get in close proximity, they can form a ring structure. By addition of ligase and polymerase, rolling circle amplification can occur. Samples were probed with fluorescence labeled oligonucleotides, complementary to the amplification product for detection of the protein interaction. PLA signals were counted from fluorescence images using ImageJ software v.1.52a (NIH).
4
Proximity Ligation Assay for PTS1 and Cyp40
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CHO-K1 cells were incubated with PT for indicated periods of time. Cells were fixed with 4% PFA, permeabilized and blocked with 10 % NGS and 1 % BSA in PBST. Subsequently, cells were incubated with mouse anti-PTS1 (Santa Cruz) and rabbit anti-Cyp40 (Thermo Fisher Scientific) antibodies for 1 h at 37 °C.
PLA assay was performed according to the manufacturer's protocol (Duolink using PLA technology, Sigma-Aldrich, Merck) and as described before 43, 44 .
PLA assay was performed according to the manufacturer's protocol (Duolink using PLA technology, Sigma-Aldrich, Merck) and as described before 43, 44 .
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