Proximity Ligation Assay to Study In Situ Protein-Protein Interactions

Samenvatting

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 interaction with DLC-1. Nematode strains co-expressing control and test proteins with 3xFLAG-tagged DLC-1 are referred to in this text as 3xFLAG::DLC-1;....

Materialen

Lijst van materialen gebruikt in dit artikel

Naam	Bedrijf	Catalogusnummer	Opmerkingen
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	4.4x8.4 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