Method Article

Bimolecular Fluorescence Complementation-Coupled Photoactivated Localization Microscopy

July 8th, 2025

In This Article

Abstract

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

Source: Nickerson, A. et al., Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM). J. Vis. Exp. (2015)

This video describes BiFC-PALM ― a combination of photoactivated localization microscopy and bimolecular fluorescence complementation ― to assess protein-protein interactions. BiFC involves the fusion of two fluorescent protein fragments with two interacting proteins of interest. When the two proteins interact, the fragments are brought into proximity, which allows the two parts to come together and reconstitute a functional fluorescent protein. The fluorescence of a single fluorophore is localized at a single molecule level inside cells by PALM.

Protocol

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,

1. Cloning

  1. Determine the configurations to clone and choose a linker. Tag proteins with the fragments on the N- or C-terminus as described below so they do not disrupt their proper localization. Use a flexible linker such as (GGGGS)x2.
  2. Genetically tag the proteins of interest to the PAmCherry1 fragments. As one option, use the cloning plasmids listed in the Materials List containing the fragments RN (PAmCherry1 residues 1-159) and RC (Met plus PAmCherry1 residues 160-236) with flanking MCS sequences.
    1. Linearize the plasmids containing the fragments RN and RC with inverse PCR (or restriction digest) at the point of insertion. Primers fo....

Access restricted. Please log in or start a trial to view this content.

Disclosures

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,
No conflicts of interest declared.

Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Microscope frameNikonTi-U
60X oil immersion TIRF objective with 1.49 NANikonCFI Apo TIRF 60x H
EMCCD cameraAndoriXon Ultra 897
561 nm laserOpto EngineMGL-FN-561
405 nm laserCoherentCUBE-405 50mW
561 nm dichroic mirrorSemrockDi01-R405/488/561/635-25x36
561 nm filterSemrockFF01-525/45-25
405/561 nm notch filterSemrockNF01-405/488/568-25
Temperature and CO2 controlled stageTokai HitINUBG2ATW-PLAM
pENTR-D-TOPO PAmCherry1_1-159-MCSAddgene60545
ENTR-D-TOPO PAmCherry160-236-MCSAddgene60546
pcDNA3.2-DESTLife Technologies12489-019
pLenti-DESTAddgenehttp://www.addgene.org/
Phusion High-Fidelity DNA PolymeraseThermo ScientificF-531
In-Fusion HD CloningClontech639649
LR ClonaseLife Technologies11791
Vira Power Lentivirus PackagingLife TechnologiesK497500
X-tremeGENE Transfection ReagentRoche13873800
Lab-Tek II Chambered CoverglassThermo Scientific155409#1.5 glass bottom dishes
U2OS cellsATCCHTB-96
293T/17 cellsATCCCRL-11268
DMEM with phenol redLife Technologies11995
DMEM no phenol redLife Technologies21063
Fetal bovine serumLife Technologies10082
Leibovit's L-15, no phenol redLife Technologies21083-027
Reduced serum mediumLife Technologies31985
Phosphate Buffered SalineLife Technologies14040
SyringeBD Biosciences309604
Syringe filterMilliporeSLHV033RB
Lentiviral concentratorClontech631231
Retroviral concentratorClontech631455
10 cm culture dishBD Biosciences353003
6-well culture plateBD Biosciences353046
PolybreneBD Biosciences353046
PuromycinLife TechnologiesA11138
G-418Calbiochem345812Neomycin
DoxycylineFisherBP2653
Tris baseFisherBP152
EDTASigmaEDS
Sodium HydroxideSigmaS5881
ParaformaldehydeSigma158127
GlutaraldehydeSigmaG6257
PIPESSigmaP6757
HEPESSigmaH4034
EGTASigma3777
Magnesium SulfateSigmaM2643
Potassium HydroxideSigma221473
Sodium chlorideFisherBP358
Magnesium chlorideFisherM33
100 nm gold particlesBBI SolutionsEM.GC100
Molecular grade waterLife Technologies10977
Dpn1New England BiolabsR0176
PCR purification kitQiagen28104
Miniprep kitQiagen27104
Midiprep kitMacherey-Nagel740410
0.6 ml microcentrifuge tubesFisher05-408-120
1.5 ml microcentrifuge tubesFisher05-408-137
15 ml tubesFisher05-539-12
5 ml polypropylene round-bottom tubesBD Biosciences352063
14 ml polypropylene round-bottom tubesBD Biosciences352059
50 ml tubeBD Biosciences352070
PCR tubeGeneMateC-3328-1
SOC mediumLife Technologies15544
LB brothBD Biosciences244610
Kanamycin sulfateFisherBP906
Competent cellsLife TechnologiesC4040

Reprints and Permissions

Request permission to reuse the text or figures of this JoVE article

Request Permission

Tags

Bimolecular Fluorescence ComplementationPhotoactivated Localization MicroscopyProtein Protein InteractionsRas Raf ComplexFluorescent Protein ReconstitutionSingle Molecule LocalizationTIRF MicroscopyEMCCD CameraGold Particle TrackingLaser Activation

Related Articles