Retroviral pBabe-puro U2OS cells stably expressing PDPK1-FLAG were plated onto coverslips pretreated with poly D-lysine. After plating, cells were treated overnight with 30μM C6 or C6nc. Cells were fixed in 4% methanol-free formaldehyde and permeabilized with 0.5% Triton. Proximity ligation assays were performed with the Duolink PLA mouse/rabbit kit (Sigma) according to the manufacturer’s instructions. Primary antibodies used were mouse anti-FLAG and rabbit anti-WDR5 (Bethyl 429A). Confocal images were acquired using an Andor DU-897 EMCCD camera mounted on a Nikon Spinning Disk Microscope.
Duolink pla mouse rabbit kit
Manufactured by Merck Group
The Duolink PLA mouse/rabbit kit is a laboratory product designed for in-situ protein-protein interaction analysis. It provides a method for detecting and visualizing the proximity of two target proteins in fixed cells or tissue samples using a proximity ligation assay (PLA) technique. The kit includes primary antibodies, oligonucleotide-conjugated secondary antibodies, and detection reagents necessary for the PLA workflow.
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Lab products found in correlation
6 protocols using duolink pla mouse rabbit kit
1
Proximity Ligation Assay for PDPK1-FLAG and WDR5
2
Proximity Ligation Assay for PCNA and HMCES
3
Proximity Ligation Assay for c-Myc and G9a
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2 × 104 cells were seeded in chamber slides 24 h prior to fixation with 4% paraformaldehyde for 30 min. Cells were subsequently permeabilised with 0.1% Triton‐X for 2 min. PLA was performed using the Duolink® PLA mouse/rabbit kit (Sigma) following the manufacturer's instructions. Briefly, cells were blocked and incubated with primary antibodies for c‐Myc (Merck Millipore) and G9a (R&D Systems, Minneapolis, MN, USA) overnight at 4 °C. Oligomer‐conjugated probes were added for 30 min at 37 °C prior to the addition of polymerase for rolling circle amplification for 100 min. Slides were mounted with Duolink® In Situ Mounting Media with DAPI. Cells were then imaged with Zeiss Imager M2 and analysed using imagej (National Institutes of Health, Bethesda, MD, USA).
4
Proximity Ligation Assay for PDPK1-FLAG and WDR5
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Retroviral pBabe-puro U2OS cells stably expressing PDPK1-FLAG were plated onto coverslips pretreated with poly D-lysine. After plating, cells were treated overnight with 30μM C6 or C6nc. Cells were fixed in 4% methanol-free formaldehyde and permeabilized with 0.5% Triton. Proximity ligation assays were performed with the Duolink PLA mouse/rabbit kit (Sigma) according to the manufacturer’s instructions. Primary antibodies used were mouse anti-FLAG and rabbit anti-WDR5 (Bethyl 429A). Confocal images were acquired using an Andor DU-897 EMCCD camera mounted on a Nikon Spinning Disk Microscope.
5
Proximity Ligation Assay for Protein Interactions
6
Proximity Ligation Assay for Protein Interactions
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Coverslips were pretreated with poly D-lysine, and then cells were plated onto coverslips at sub-confluence. Cells were fixed in 4% methanol-free formaldehyde for ten minutes and permeabilized with 0.5% Triton for ten minutes. Proximity ligation assays were performed with the Duolink PLA mouse/rabbit kit (Sigma) according to the manufacturer’s instructions. Nuclei were stained using Duolink in situ mounting medium with DAPI (Sigma DUO82040). Primary antibodies used were mouse anti-MYC (Santa Cruz c33) and rabbit anti-BAF155 (Abcam ab72503). Confocal images were acquired using an Andor DU-897 EMCCD camera mounted on a Nikon Spinning Disk Microscope. Fluorescent puncta were quantified by using FIJI (ImageJ) to calculate the number of maxima within each nucleus.
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