Proximity Ligation Assay for Protein-Protein Interactions

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 before^{46 (link),47 (link)}.

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Ernst K., Mittler A.K., Winkelmann V., Kling C., Eberhardt N., Anastasia A., Sonnabend M., Lochbaum R., Wirsching J., Sakari M., Pulliainen A.T., Skerry C., Carbonetti N.H., Frick M, & Barth H. (2021). Pharmacological targeting of host chaperones protects from pertussis toxin in vitro and in vivo. Scientific Reports, 11, 5429.

Publication 2021

mouse anti-PTS1 rabbit anti-Cyp40 Duolink using PLA technology

Antibodies Assay Cells Cho cells Fkbp51 Hsp70 Hsp90 Mouse Rabbit

Corresponding Organization : Universität Ulm

Other organizations : University of Turku, University of Maryland, Baltimore

Top 5 similar protocols

Variable analysis

independent variables

Incubation time with PT

dependent variables

Interactions between PTS1 and Cyp40, Hsp90, Hsp70, or FKBP51

control variables

CHO-K1 cells
4% PFA fixation
10% NGS and 1% BSA in PBST for permeabilization and blocking
Primary antibodies (mouse anti-PTS1, rabbit anti-Cyp40, rabbit anti-Hsp90, rabbit anti-Hsp70, rabbit anti-FKBP51)

controls

Positive control: Not specified
Negative control: Not specified

Annotations

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