Here we describe the basic protocol for using the commercially available Proximity Ligation Assay materials (Sigma〢ldrich, St. Louis, MO), and incorporate details to aid the researcher in successfully performing the experiments. 2020 Wiley Periodicals LLC. Basic Protocol 1: Proximity ligation assay ...
PLA was carried out using either the Duolink PLA kits for brightfield (Sigma, #DUO92012) or fluorescent applications (Sigma, #DUO92008), NaveniBright HRP (Navinci, #NB.MR.HRP.100) for brightfield applications, or a custom-made detection kit utilising biotin in the fluorophore labelling proc...
Proximity ligation assay (PLA)26 or proximity extension assay (PEA)27, which record the colocalization of two probes by ligating or extending probe-bound DNA sequences, now routinely multiplex ~100 signals with orthogonal sequences. The information content of DNA itself is much larger, however. ...
Dextran sulfate sodium salt from Sigma. Hard to dissolve – keep aliquots at 4C for months. Mouse Cot-1 DNA: Invitrogen cat # 18440-016 Human Cot-1 DNA: Invitrogen cat # 15279-011 Dry slides as much as possible to avoid diluting probes. ...
(1:10, Sigma-Aldrich) or anti-goat MINUS (1:10, Sigma-Aldrich) at 37 °C for 1 h to detect primary antibody pairs positioned in proximity. After washes, the wells were incubated with 40 μl ligation mix (0.05 unit/μl T4DNA ligase(ThermoFisher), 250 μg/mlbovine serum albumin(BSA...
Duolink proximity-ligation assay The endogenous interaction of Rab5 and Gapex5 was observed using a Duolink kit (Sigma, St. Louis, MO, USA), as described by the manufacturer. Briefly, cells were incubated with mouse anti-Rab5 and rabbit anti-Gapex5 antibodies and then incubated with proxim...
Proximity ligation assay is an “in situ” detection method for protein-protein interactions. Fluorescence PLA was performed according to manufacturer protocol (Duolink In Situ, Red, Mouse/Rabbit, SigmaAldrich) with modifications for brain tissue samples as follows. Brain tissue slides were deparaffin...
Using this assay, the antibody used herein has been shown to detect Aβ protofibrils also in other, well-known, tg-mice such as PSAPP and tg2576 [11]. Here, we demonstrate that the proximity ligation assay (PLA) can provide even more sensitive detection of synthetic Aβ protofibrils. PLA...
(pTyr): Tyrosine phosphorylation (PLA): Proximity ligation assay (PTM): Post translational modification amplification (RCA): Rolling circle (TMA): Tissue micro array (MS): Mass spectrometry.References Zwick E, Bange J, Ullrich A: Receptor tyrosine kinase signalling as a target for cancer...