Team:Slovenia/METHODS and PARTS/protocols/sfm

Fun fact:

Binding assays in mammalian cells (Cell cultures)

In our experiments we used human embryonic kidney cell line HEK293. Cells were grown in DMEM medium containing 10% FBS. Transfection was performed using jetPEI transfection reagent which was used according to manufactuer's protocol. Cells were transfected with the desired plasmids and grown until assaying. For microscopy, cells were tipically seeded in slide chambers, whereas for fluorimetry and split-fusion protein production, where larger numbers of cells were needed, cells were grown in 6 well plates.

Fluorimeter

We used Perkin Elmer instruments LS55 Luminescence Spectrometer to measure fluorescence intensities of cell lysates. Fluorescence spectra were obtained according to procedures in the literature. mCerulean and mCitrine were excited at 433 and 516 nm, respectively. For FRET tests, emission spectra was measured after samples were excited samples with 433 nm. The expected additonal peak at 529 nm indicating presence of FRET effect was observed. During spectral scans we adjusted some parameters including voltage of the photomultiplier and slit width as well as the speed of spectral scans (usually 200 nm per minute) in order to improve aquired spectra. We also used BioTek's Synergy MX spectrometer to allow simultaneous fluorescence scan measurements of up to 96 samples. Running experiments on this machine alowed us to follow gradual increase of fluorescence signal as a result of reconstitution of split fluorescent proteins with zinc finger domains followed by their binding to DNA program.

Confocal microscopy

Leica TCS SP5 laser confocal microscope with inverted microscope was used for in vivo detection of split fluorescent proteins on a DNA program and FRET between mature GFPs. Transfected HEK293 cells were cultured overnight on 8-well microscope slide at 37°C. For observation an 63 X oil immersion objective was used. Cells were excited at 433 nm for mCerulean and 516 nm for mCitrine. FRET was detected according to the methodology provided by the manufacturer's software (FRET AB and FRET SE wizards within Leica LAS AF computer software).

Split-FRET experiments

HEK293 cells were transfected with mammalian expression vectors carrying split fluorescent fusion proteins under CMV promoter using jet PEI tranfection reagent protocol and ultimately fixed with 4% paraformaldehyde. Split fluorescent protein reconstitution as well as FRET effect were observed under confocal microscope (described under Confocal microscopy). mCerulean (Gli1_linker_peptide – nCFP and cCFP_linker peptide_ ZNF HIVC) and mCitrine (PBSII_linker peptide_nYFP and cYFP_linker peptide_Zif268) were reconstituted only in the presence of specific, but not random, DNA program. Split fluorescent proteins bind to adjacent target nucleotide sequences when plasmids carrying split fluorescent protein fusions are cotransfected with a plasmid carrying target DNA program . As split fluorescent proteins cannot form a fully formed chromophore in vivo by accident, fluorescent signal is a consequence of split protein – zinc finger fusions bound to a DNA program sequence.