Bimolecular Fluorescence Complementation-Coupled Photoactivated Localization Microscopy

Published: May 31, 2023

Abstract

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

1. Cloning 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. 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…

Declarações

The authors have nothing to disclose.

Materials

Microscope frame Nikon Ti-U
60X oil immersion TIRF objective with 1.49 NA Nikon CFI Apo TIRF 60x H
EMCCD camera Andor iXon Ultra 897
561 nm laser Opto Engine MGL-FN-561
405 nm laser Coherent CUBE-405 50mW
561 nm dichroic mirror Semrock Di01-R405/488/561/635-25×36
561 nm filter Semrock FF01-525/45-25
405/561 nm notch filter Semrock NF01-405/488/568-25
Temperature and CO2 controlled stage Tokai Hit INUBG2ATW-PLAM
pENTR-D-TOPO PAmCherry1_1-159-MCS Addgene 60545
ENTR-D-TOPO PAmCherry160-236-MCS Addgene 60546
pcDNA3.2-DEST Life Technologies 12489-019
pLenti-DEST Addgene http://www.addgene.org/
Phusion High-Fidelity DNA Polymerase Thermo Scientific F-531
In-Fusion HD Cloning Clontech 639649
LR Clonase Life Technologies 11791
Vira Power Lentivirus Packaging Life Technologies K497500
X-tremeGENE Transfection Reagent Roche 13873800
Lab-Tek II Chambered Coverglass Thermo Scientific 155409 #1.5 glass bottom dishes
U2OS cells ATCC HTB-96
293T/17 cells ATCC CRL-11268
DMEM with phenol red Life Technologies 11995
DMEM no phenol red Life Technologies 21063
Fetal bovine serum Life Technologies 10082
Leibovit's L-15, no phenol red Life Technologies 21083-027
Reduced serum medium Life Technologies 31985
Phosphate Buffered Saline Life Technologies 14040
Syringe BD Biosciences 309604
Syringe filter Millipore SLHV033RB
Lentiviral concentrator Clontech 631231
Retroviral concentrator Clontech 631455
10 cm culture dish BD Biosciences 353003
6-well culture plate BD Biosciences 353046
Polybrene BD Biosciences 353046
Puromycin Life Technologies A11138
G-418 Calbiochem 345812 Neomycin
Doxycyline Fisher BP2653
Tris base Fisher BP152
EDTA Sigma EDS
Sodium Hydroxide Sigma S5881
Paraformaldehyde Sigma 158127
Glutaraldehyde Sigma G6257
PIPES Sigma P6757
HEPES Sigma H4034
EGTA Sigma 3777
Magnesium Sulfate Sigma M2643
Potassium Hydroxide Sigma 221473
Sodium chloride Fisher BP358
Magnesium chloride Fisher M33
100 nm gold particles BBI Solutions EM.GC100
Molecular grade water Life Technologies 10977
Dpn1 New England Biolabs R0176
PCR purification kit Qiagen 28104
Miniprep kit Qiagen 27104
Midiprep kit Macherey-Nagel 740410
0.6 ml microcentrifuge tubes Fisher 05-408-120
1.5 ml microcentrifuge tubes Fisher 05-408-137
15 ml tubes Fisher 05-539-12
5 ml polypropylene round-bottom tubes BD Biosciences 352063
14 ml polypropylene round-bottom tubes BD Biosciences 352059
50 ml tube BD Biosciences 352070
PCR tube GeneMate C-3328-1
SOC medium Life Technologies 15544
LB broth BD Biosciences 244610
Kanamycin sulfate Fisher BP906
Competent cells Life Technologies C4040

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Bimolecular Fluorescence Complementation-Coupled Photoactivated Localization Microscopy. J. Vis. Exp. (Pending Publication), e21410, doi: (2023).

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