PLA was carried out with Duolink® In Situ Detection Reagents Orange (#DUO92007, Sigma Aldrich). Additional reagents Duolink® In Situ PLA® Probe Anti-Rabbit PLUS/MINUS (#DUO92002/DUO92005, Sigma Aldrich) and Duolink® In Situ PLA® Probe Anti-Mouse PLUS/MINUS (#DUO92001/DUO92004, Sigma Aldrich) were used. Optimization of manufacturer’s procedure applied to non-adherent cells is the goal of this article and is described underneath. One representative experiment of at least three independent experiments is shown.
Duolink in situ pla probe anti rabbit plus minus
Manufactured by Merck Group
The Duolink® In Situ PLA® Probe Anti-Rabbit PLUS/MINUS is a laboratory equipment product. It is used for in situ proximity ligation assay (PLA) applications. The product consists of a pair of oligonucleotide-labeled secondary antibodies that bind to a primary antibody targeting a specific protein.
Automatically generated - may contain errors
2 protocols using duolink in situ pla probe anti rabbit plus minus
1
Optimized Duolink® PLA for Suspension Cells
2
Quantifying Protein-Protein Interactions
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For PLA experiments (68 (link)) with SLP65, pSLP65, RHOA and GEF-H1 the corresponding antibodies were used (RHOA (26C4) santa cruz sc-418; BLNK (2B11) santa cruz sc-8003; Phospho-BLNK (Thr152) (E4P2P) Cell Signaling #62144; GEF-H1 (55B6) Cell Signaling #4076). The antibodies for RHOA and BLNK were labeled directly using the Duolink in situ Probemaker plus/minus reagent (Sigma-Aldrich). GEF-H1 and pSLP65 were detected by the corresponding secondary antibody Duolink in situ PLA Probe Anti-Rabbit plus/minus (Sigma-Aldrich).
The PLA probes were then subjected to ligation and polymerization reactions using the Duolink in situ detection reagents orange (Sigma-Aldrich). In the end, the cells were examined for the frequency of signals per cell under the fluorescence microscope (Leica). Pictures were taken and quantified by Image J software.
The PLA probes were then subjected to ligation and polymerization reactions using the Duolink in situ detection reagents orange (Sigma-Aldrich). In the end, the cells were examined for the frequency of signals per cell under the fluorescence microscope (Leica). Pictures were taken and quantified by Image J software.
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