Proximity Ligation Assay (PLA) Protocol

Introduction

PLA may be used in two formats:

• Fixed cells/tissues mounted on slides (immunofluorescence - IF)
• Fixed cells in solution (flow cytometry or co-immunoprecipitation)

The following protocol covers the IF format which is provided for guidance purposes. A series of optimization steps will be critical to ensure a positive outcome.

Oligo Considerations

As well as ordering antibody-oligo conjugates for use in PLA from AbOliGo, additional oligos are required for the experiment. Sequence and function of each oligo is provided below:

Protocol

A. Cell Fixation + Permeabilization

1. Fix cells with 4% PFA in PBS, 15 min RT.
2. Wash 3× with PBS.
3. Permeabilize with 0.5% Triton X-100 in 1% BSA/PBS, 30 min at 37 °C.
4. Wash 3× with PBS.

B. PLA Binding (Antibody-Oligo Conjugates, AOCs)

1. Keep cells covered with a 2–5 µL liquid film so they never dry.
2. Add antibody-oligo conjugates at 2–25 ng/µL, 1 h at RT (optimal dilution will have to be pre-determined for each antibody-oligo conjugate).
3. Wash 4× with TBS-T over a total of 1 h.

C. Connector Hybridization + Ligation (Circle Formation)

Incubate 45 min at 30 °C in 1× T4 ligase buffer containing:

• T4 DNA ligase: 1 U/µL
• Connector strands: 125 nM (Connector 1 + Connector 2)
• BSA: 125 ng/mL
• ddH₂O to volume

Wash 3× with TBS-T.

D. Rolling Circle Amplification (RCA)

Incubate 100 min at 32 °C (orbital shaking) in 1× phi29 buffer containing:

• phi29 polymerase: 0.25 U/µL
• dNTPs: 200 µM
• BSA: 200 µg/mL

Wash 2× with TBS-T.

E. Fluorescent Detection

Counterstain 30 min in SSC containing:

• DAPI: 1 µg/mL
• Detection probe: 6 nM
• Phalloidin-Atto488: 20 nM

Wash 3× with TBS, then image by fluorescent microscope.

Hints and Tips

1. Use Controls That Actually Diagnose Which Stage of the PLA is Failing

PLA has multiple "gates" (antibody binding → connector hybridization → ligation → RCA → detection probe hybridization). Use relevant controls to confirm each stage has worked:

2. Don't Over-Interpret "Puncta" as "Direct Binding"

PLA is fundamentally a proximity readout. Although the technique is powerful, a positive dot can also occur if proteins are packed tightly in a busy area of the cell, or if the antibodies sit in a way that brings the DNA probes close, even when the proteins aren't truly interacting.

What to do:

• Confirm with orthogonal evidence (co-IP, FRET/BRET, genetic epistasis) when the claim is "direct interaction"
• Use biological controls (KO/knockdown, mutation that breaks binding, ligand dependence)

3. Oligo 5' and 3' Considerations

Ligation at a nick requires the right end chemistry (classically 5' phosphate / 3' hydroxyl at the ligation junction). If you change suppliers or reorder oligos, confirm whether the connector oligos are ordered phosphorylated at the 5' end. This is a common "everything binds but no circles form" failure mode.

4. Sample Handling

• Fixation/permeabilization/antigen retrieval must be compatible for both AOCs; optimize each individually using primary and the relevant secondary antibodies. Then combine for the PLA assay.
• Too-high AOC concentrations are a classic reason for high background; titrate each AOC individually while holding the other constant if you are having background issues.
• Never allow the samples to dry out, best to use a humidifying chamber.
• Remove residual wash buffer before ligation/amplification with Kimwipes; leftover wash dilutes enzymes and reduces efficiency.
• Prevent cross-contamination between samples, wash different antibody conditions separately when possible; avoid bulk washing when multiple antibody pairs/controls are on the same run.
• Keep enzymes cold while setting up; don't let diluted enzyme mixes sit around.

Troubleshooting

References

• Preparation of single- and double-oligonucleotide antibody conjugates and their application for protein analytics
• The method developer's guide to oligonucleotide design