In vitro applications of GFP Pallette of fluorescent

Fluorescent proteins are versatile tool for multicolor microscopic imaging but can they be used differently? Common Applications

• Localization • dynamics – Diffusion – Active relocation All at the level of the microscope • Protein interactions • Single molecule studies • etc Can GFP be used for non-microscopic applications?

YES, GFP is a tag GFP as tag for

• What is immunoprecipitation? – By immunoprecipitation a protein of interest is precipitated by means of specific – Antibody is linked to a mass structure (e.g., an agarose microbead) – Other proteins interacting with the protein of interest may be pulled down during IP assay (co-IP)

GFP as tag for immunoprecipitation

Original immunoprecipitation recipes required radioactive labeling GFP as tag for immunoprecipitation

• Many commercial exist for protein of interest. But what if not? – If not, protein of interest can be tagged – Tags may be small peptides and proteins (e.g., polyHis, FLAG, GST, Myc but also FPs) – All tags except FPs cannot be detected in living cells GFP as tag for immunoprecipitation

• FLAG: octapeptide DYKDDDDK • Myc: a peptide derived from the sequence of cMyc transcription factor (N-EQKLISEEDL-C) • 6×His: a polyhistidine tag that rarely occurs in native proteins. Does not need antibody for pull down. Can do so with Ni-NTA (nickel-nitrilotriacetic acid) • GST: tag corresponding to glutathione S transferase enzyme (binds to reduced form of GSH; glutathione) GFP as tag for immunoprecipitation

• Common problem of immunoglobulins is there multimeric structure • When the product of Co-IP will be denatured it will be contaminated by LCs and HCs GFP as tag for immunoprecipitation

Contamination by HCs and LCs may hinder our protein of interest GFP as tag for immunoprecipitation

Use of camelid antibodies GFP as tag for immunoprecipitation

Camelid antibodies resolve the problem of contamination GFP as tag for immunoprecipitation

IP: LER IP: MPK6 IN IP IN IP

MPK6-GFP

MAP65-1 has similar weight to HCs and it would had been missed MAP65-1 Protein-protein interactions in vitro

• Protein-protein interactions are only identified by Co-IP but not quantified • Quantifications of PPI can give information of

– Kds – Interaction kinetics Protein-protein interactions in vitro

• Surface plasmon resonance • Isothermal titration calorimetry • Forster resonance energy transfer Protein-protein interactions in vitro

SPR relies on the evanescent wave produced under total internal reflection Protein-protein interactions in vitro

Too expensive, labor intensive, prone to artefacts Protein-protein interactions in vitro

• Based on thermodynamics of interactions and heat exchange between reactants (endo- vs. exothermic reactions) • We measure temperature changes due to interactions Protein-protein interactions in vitro

Very accurate and real-time. Needs huge amounts of reactants very specialized equipment and expertise Protein-protein interactions in vitro Flow cytometry and sorting

Separation of cells from a complex population based on differential fluorescence labeling Flow cytometry and cell sorting

After excitation fluorophores become differentially charged and can be separated by electromagnetic fields Flow cytometry and cell sorting

Before FPs, flow cytometry could be only used in fixed immunolabeled cells Flow cytometry and cell sorting

• By applying GFP technology and knock-in gene targeting, selective and rare cell populations (e.g., cells of the immune system) can be specifically isolated, subcultured and further studied Subcellular fractionation

• GFP-tags are normally targeted to subcellular organelles • We can used differentially labeled cells to obtain and characterize homogeneous populations of organelles Subcellular fractionation

Straight forward detection of fractions In vivo bioimaging

GFP tagging can be used to tag entire organs of biomedical important models In vivo bioimaging In vivo bioimaging

• By selective gene targeting we can tag cancer markers • We can use specialized MRI or CT scans to locate tumors with high precision • Tumors can then be specifically eradicated by directed photo-damage with focused laser beams