Proximity Ligation Assay to Study In Situ Protein-Protein Interactions
Published: May 31, 2023
Abstract
Source: Wang, X., et al., In Situ Detection of Ribonucleoprotein Complex Assembly in the C. elegans Germline using Proximity Ligation Assay. J. Vis. Exp. (2020)
This video describes the proximity ligation assay to study in situ protein-protein interactions in nematode cells. The close proximity of the proteins of interest generates the circular DNA template with the help of a PLA probe attached to a secondary antibody. The probe is further amplified using the amplification solution, which produces a long single-stranded DNA followed by the hybridization of the detector probe, enabling the localization of protein-protein interactions in cells.
Protocol
NOTE: This protocol uses C. elegans strains in which potential interacting partners are both tagged. It is strongly recommended that a negative control strain be used, in which one tagged protein is not expected to interact with another tagged candidate interaction partner. Here, GFP alone was used as a negative control to assess background, as DLC-1 is not expected to interact with GFP in the worm. GFP-tagged OMA-1 was used as the experimental strain, as preliminary data suggest an in…
Açıklamalar
The authors have nothing to disclose.
Materials
| 16% paraformaldehyde solution | Electron Microscopy services | 15710 | Used to make 4% working solution |
| 1M KH2PO4 | Sigma | P0662 | Prepare a 1M working stock |
| 1x M9 | Various | various | Prepared as 10x stock used at 1x; see wormbook.org for protocol |
| 1x PBS | Various | various | see wormbook.org for protocol |
| 26.5-Gauge Needle | Exel International | 26402 | Needles used for dissection |
| BSA | Lampire | 7500802 | |
| Centrifuge Tubes | Thermo Scientific | 05-529C | 50ml Oak ridge centrifuge tube used for synchronization |
| Confocal Microscope | Zeiss | 880 | |
| Coplin Jar | PolyLab | 62101 | |
| Coverslip to Freeze Sample | Globe Scientific | 1411-10 | 22x40mm, No. 1 |
| Coverslip to Seal Slide | Globe Scientific | 1404-15 | 22x22mm, No. 1.5 |
| DAPI Mounting Medium for Immunofluorescence | Vector | H-1200 | |
| Ligase | Sigma-Aldrich | DUO82029 | Duolink 1x Ligase, Comes as part of the Duolink In Situ Detection Reagents Red kit DUO92008 |
| Amplification red buffer | Sigma-Aldrich | DUO82011 | Duolink 5x Amplification Red buffer, Comes as part of the Duolink In Situ Detection Reagents Red kit DUO92008 |
| Ligation Buffer | Sigma-Aldrich | DUO82009 | Duolink 5x Ligation buffer, Comes as part of the Duolink In Situ Detection Reagents Red kit DUO92008 |
| Antibody Diluent | Sigma-Aldrich | DUO82008 | Duolink antibody diluent,Comes with DUO92004 and DUO92002, Note: A 1x PBS/1% BSA solution can also be used as a substitute to dilute the antibody. |
| Blocking Solution | Sigma-Aldrich | DUO82007 | Duolink blocking solution, Comes with DUO92004 and DUO92002 |
| Mounting Medium for PLA | Sigma-Aldrich | DUO82040 | Duolink In Situ mounting medium with DAPI |
| MINUS Probe | Sigma-Aldrich | DUO92004 | Duolink In Situ Probe Anti-Mouse MINUS |
| PLUS Probe | Sigma-Aldrich | DUO92002 | Duolink In Situ Probe Anti-Rabbit PLUS |
| Wash Buffer A | Sigma-Aldrich | DUO82046 | Duolink In Situ wash Buffer A |
| Wash Buffer B | Sigma-Aldrich | DUO82048 | Duolink In Situ wash Buffer B |
| Polymerase | Sigma-Aldrich | DUO82030 | Duolink Polymerase, Comes as part of the Duolink In Situ Detection Reagents Red kit DUO92008 |
| Epifluorescent Microscope | Leica | DFC300G camera, DM5500B microscope | |
| Goat anti-mouse Alexa 594 | JacksonImmuno | 115-585-146 | Use at 1:500 |
| Goat anti-rabbit Alexa 488 | JacksonImmuno | 111-545-144 | Use at 1:200 |
| Glass Pipette | Corning | 7095B-5X | |
| Levamisole | ACROS Organics | 187870100 | Prepare a 250mM working stock |
| Methanol | Fisher Scientific | A454 | |
| Mouse anti-FLAG | Sigma | F1804 | Use at 1:1000 for immunofluorescence and PLA, pre-block with normal goat serum recommended |
| Nailpolish | L.A. colors | CNP195 | |
| Nematode Growth Medium (NGM) | Various | See wormbook.org for protocol | |
| Normal Goat Serum | JacksonImmuno | 005-000-121 | |
| Polyethylene Pasteur Pipette | Globe Scientific | 135030 | |
| Poly-L-Lysine | Sigma-Aldrich | P1524 | Prepared as 0.1% stock solution in water, stored at -20C, and diluted 1:100 in water to coat slides |
| Petri Dishes | Tritech | PD7060 | 60 mm diameter |
| Rabbit anti-GFP | Thermo Fisher | G10362 | Use at 1:200 for immunofluorescence, 1:4000 for PLA |
| Slides | Thermo Fisher | 30-2066A-Brown | Three-square 14x14mm autoclavable slides with bars are custom-ordered through Fisher Scientific. Poly-L-Lysine added to slides in the lab |
| Sodium Hypochlorite solution | Fisher Scientific | SS290-1 | |
| Task wipes | Kimtech | 34120 | 4.4×8.4 inch task wipes |
| Trays (242x241x20mm) | Thermo Fisher | 240845 | Used to make humid chamber |
| Triton X-100 | ACROS Organics | 327372500 | |
| Ultrapure water | Milli-Q | Ultrapure water obtained from Milli-Q Integral Water Purification System | |
| Watchglass | Carolina Biological | 742300 | |
| -20 °C freezer | |||
| -80 °C freezer | |||
| Aluminum Foil | |||
| OP50 strain E. coli | |||
| Orbital Shaker | |||
| Tape | |||
| Nematode strains used in this study (both available upon request) | |||
| mntSi13[pME4.1] II; unc-119(ed3) III; teIs1 [pRL475] | UMT 376 | dlc-1 prom::3xFLAG::dlc-1::dlc-1 3'UTR; oma-1 prom::oma-1::GFP; Reference 24 | |
| mntSi13[pME4.1] II; mntSi21[pXW6.22] unc-119(ed3) III | UMT 422 | dlc-1 prom::3xFLAG::dlc-1::dlc-1 3'UTR; gld-1 prom::ceGFP::fbf-1 3'UTR + unc-119(+); Reference: this study |
Etiketler
Bu Makaleden Alıntı Yapın
Proximity Ligation Assay to Study In Situ Protein-Protein Interactions. J. Vis. Exp. (Pending Publication), e21396, doi: (2023).