This study was funded by: National Research Network on Aging (NFN S93) by the Austrian Science Foundation (FWF), European Commission Integrated Projects MiMAGE and PROTEOMAGE, Netherlands Genomics Initiative/Netherlands Organization for Scientific Research (NGI/NWO; 05040202 and 050-060-810 NCHA), the EU funded Network of Excellence Lifespan (FP6 036894), and Innovation Oriented Research Program on Genomics (SenterNovem; IGE01014 and IGE5007).

1. Plasmid Construction
<ol>
 <li>Order custom oligo-nucleotides encoding for dgn (ACKNWFSSLSHFVIHL11) and for dgnFS (HARTGSLACPTSSSICE) and ligate it into the pEGFP-C1 vector to obtain the fusion of the GFP with dgn/dgnFS (Figure 1). </li>
 <li>Amplify the coding sequence for GFP-dgn and GFP-dgnFS by PCR according to the protocol of the pENTR Directional TOPO Cloning Kit and continue with the pLenti6/V5 Directional TOPO Cloning Kit (Figure 6). </li>
</ol>
2. Virus Production
<ol>
 <li>The day before transfection (Day 1) seed out HEK 293FT cells in a T75 flasks so that they will be 90-95% confluent on the day of transfection. </li>
 <li>On the day of transfection dilute 36 &mu;l of lipofectamine 2000 Reagent in 1.5 ml of DMEM without serum and antibiotics in a 15 ml falcon. Use another 15 ml falcon and dilute 3 &mu;g pLenti6/V5 carrying the complementary DNA construct for either GFP-dgn or GFP-dgnFS, 2.5 &mu;g envelope encoding plasmid pMD2.G and 7.5 &mu;g vector backbone psPAX2 in 1.5 ml of DMEM without serum and antibiotics. After 5 min the diluted DNA is combined with the diluted lipofectamine 2000 Reagent. </li>
 <li>Incubate the mixture for 20 min at room temperature to allow the DNA-lipofectamine complexes to form. </li>
 <li>Remove the medium of HEK 293FT cells, replace it carefully by 7 ml of medium (without antibiotics) and add the DNA-lipofectamine mixture to the flask and rock it back and forth for mixing (Day 2). Incubate the flask overnight at 37 &deg;C in a humidified 5% CO2 incubator. </li>
 <li>Change medium the next day (10 ml medium without antibiotics; Day 3). </li>
 <li>After 48 hr (Day 5) harvest supernatant, centrifuge at 300 x g for 5 min at room temperature and filter the supernatant through a 0.45 &mu;m PVDF filter. </li>
</ol>
Culture medium - DMEM (HEK 293FT)
DMEM 
10% FBS 
0.1 mM MEM NEAA 
6 mM L-glutamine 
1 mM MEM Sodium Pyruvate 
1% Pen-Strep (optional) 
500 &mu;g/ml Geneticin (optional)
3. Virus Concentration by Polyethylene Glycol (PEG) Precipitation
<ol>
 <li>Add one volume Polyethylene glycol solution (50 mM Polyethylene glycol, 41 mM NaCl, autoclave, pH =7.2; PEG) to four volumes of supernatant and incubate for 2 hr at 4 &deg;C. Carefully mix it every 20-30 min by inverting. </li>
 <li>Spin down at 1500 x g for 30 min at 4 &deg;C. A white pellet should be visible. </li>
 <li>Aspirate the supernatant and centrifuge again at 1500 x g for 5 min at 4 &deg;C. Aspirate the remaining PEG solution. </li>
 <li>Resuspend the pellet in medium or phosphate-buffered saline (PBS) by pipetting up and down and vigorously vortex for 20 to 30 seconds. As a guideline use 500 &mu;l for one T75 flask and aliquot it to 100 &mu;l. Store the virus at -80 &deg;C. </li>
</ol>
4. Titering of Virus
<ol>
 <li>Seed out 5&times;104 U2-OS cells to each well of a 6-well plate the day before transfection. </li>
 <li>On the day of transfection remove the culture medium and replace it by 1 ml of DMEM containing 8 &mu;g/ml polybrene (hexadimethrine bromide). Dilute the concentrated virus supernatant 1:10 and 1:100 and add 1 &mu;l each to one well. For higher virus concentrations use 1 &mu;l, 5 &mu;l and 10 &mu;l of concentrated virus supernatant for the remaining wells. One well is left as a positive control. </li>
 <li>The following day replace the medium by 2 ml of DMEM. </li>
 <li>Start with the selection the next day. Apply 10 &mu;g/ml Blasticidin on each well. Replace the antibiotic containing medium every second day. </li>
 <li>Approximately 6-7 days after starting the selection the untransduced cells are dead. For the staining wash the cells three times with PBS and cover the cells with crystal violet (10 mg/l crystal violet in 20% ethanol) and incubate for 5-10 min at room temperature. Aspirate crystal violet (can be reused a few times) and rinse the wells twice with ddH2O and dry the plate. </li>
 <li>Count the colonies and multiply with 1,000 and the corresponding dilution (Figure 7). This procedure allows to calculate transfection units (TU) of the virus / ml. </li>
</ol>
Culture medium - DMEM (HFF-2/U2-OS)
DMEM 
10% FBS 
6mM L-glutamine 
1% Pen-Strep
5. Transduction of Human Diploid Fibroblasts (This procedure can be used for any cell type.)
<ol>
 <li>Seed out 5&times;104 human diploid fibroblasts (HDFs) to 6-well plates the day before transduction. </li>
 <li>Use a multiplicity of infection of two together with 8 &mu;g/ml polybrene as transduction enhancer in a total of one milliliter. </li>
 <li>Change medium the next day. </li>
 <li>When the cells are at 70-80 % of confluency start the selection with 10 &mu;g/ml blasticidin. After the selection is completed (no more untransfected cells die) the expansion of the cells can start and the cells are ready for experiments. Chronic selection with blasticidin allows to generate a stable cell line overexpressing GFP-dgn or GFP-dgnFS fusion proteins, ready to measure proteasome activity. This procedure guarantees a very high transfection efficiency independent of the cell type. </li>
</ol>
6. Measurement by Flow Cytometry
<ol>
 <li>Seed out 1&times;105 cells (carrying either GFP-dgn or GFP-dgnFS) the day before the experiment. </li>
 <li>Treat control cells 3 hr prior the measurement with the proteasome inhibitor, e.g. 100 &mu;g/ml LLnL.</li>
 <li>Wash twice with PBS and harvest the cells using 0.5 ml of trypsin. To stop the trypsinisation use 4.5 ml of culture medium and transfer the cells to a 15 ml falcon. </li>
 <li>Spin the cells at 300 x g for 5 min at room temperature. </li>
 <li>Aspirate the medium, resuspend the cells in PBS and spin down again at 300 x g for 5 min at 37 &deg;C. </li>
 <li>Aspirate the PBS, resuspend in 400 &mu;l of FACS buffer (10 mM Hepes, 140 mM NaCl, 2.5 mM CaCl2, pH=7.4) and transfer the cells into FACS tubes. </li>
 <li>Keep the cells on ice and measure samples by flow cytometry. The cells should not be stored longer than 30 min at 4 &deg;C. </li>
</ol>
7. Representative Results
The GFP-dgn fusion protein carries a sequence which is targeted to the proteasome and therefore the protein is immediately degraded; it corresponds to the decrease in GFP fluorescence signal. The frameshift mutant (GFP-dgnFS) carries a mutated version of this sequence and is not degraded by proteasome; it leads to the higher green fluorescence. For these reasons, young HDFs with expected high proteasome activity and transduced with GFP-dgn show a low (6% positive cells) fluorescence signal in both flow cytometry measurement and in epifluorescence (Figure 2A and B, Figure 3). The same HDFs transduced with GFP-dgnFS display 39.7% of positive cells. The treatment of the cells with proteasome inhibitor LLnL (N-acetyl-L-leucyl-L-leucyl-L-norleucinal) raised the signal to maximum of 62.9% in both, GFP-dgn and GFP-dgnFS cells (Figure 2A and B).
To determine possible decline in proteasome activity in aged human skin samples, dermal fibroblasts isolated from young, middle-aged and old donors were infected with GFP-dgn and GFP-dgnFS, as described above and cultivated to the same passage number before analysis by flow cytometry. In these experiments, a clear-cut increase in GFP signal between young individuals (11.2&plusmn;0.88% GFP-positive cells) and middle-aged donors (20.4&plusmn;2.27% GFP-positive cells, P=0.003) has been observed, indicating a decrease in proteasome activity in samples obtained from aged donors7 (Figure 4). No further decrease in proteasome activity was observed in fibroblasts isolated from oldest individuals (Figure 4). Fluorescence intensity for GFP-dgnFS was in all cases ~90% (data not shown) which indicates a high transfection efficiency for the three different age groups.
<img src="/files/ftp_upload/3327/3327fig1.jpg" alt="Figure 1" /> 
Figure 1. The GFP-dgn and GFP-dgnFS sequence. Displayed is the 3'-end of GFP (green) the multiple cloning site of the pEGFP-CL1 vector (grey) and the dgn/dgnFS sequence (red).
<img src="/files/ftp_upload/3327/3327fig2.jpg" alt="Figure 2" /> 
Figure 2. Flow cytometry analysis of GFP-dgn and GFP-dgnFS human diploid fibroblasts. A. Young human foreskin fibroblasts (HFF-2) were infected with lentiviral vectors carrying a green fluorescent protein (GFP)&#x2013;dgn gene or a GFP&#x2013;dgnFS (frameshift) construct, as indicated and analyzed for GFP fluorescence using flow cytometry (FACS Canto II, Becton Dickinson). Where indicated, cells were also treated for 3h with proteasome inhibitor N-acetyl-L-leucyl-L-leucyl-L-norleucinal (LLnL). Uninfected cells were used as a control. Experiments were performed in triplicates. B. Data are representative of three independent experiments. The numbers reflect the amount of GFP positive cells. <a href="https://www.jove.com/files/ftp_upload/3327/3327fig2large.jpg" target="_blank"> Click here to view larger figure</a>.

<img src="/files/ftp_upload/3327/3327fig3.jpg" alt="Figure 3" /> 
Figure 3. Fluorescence microscopy analysis of GFP-dgn and GFP-dgnFS HFF-2 cells. 
Young HFF-2 were treated as in Figure 2. At 9 days after infection, cells were visualized by fluorescence and phase contrast microscopy.
<img src="/files/ftp_upload/3327/3327fig4.jpg" alt="Figure 4" /> 
Figure 4. Changes in proteasome activity in human skin aging. 
The human foreskin fibroblasts from nine different donors in the indicated age groups were minimally expanded and infected with lentiviral constructs coding for GFP&#x2013;dgn. At 9 days after infection, the cells were analyzed by flow cytometry. Data were obtained on three samples per age group in duplicates (&plusmn; SE).
<img src="/files/ftp_upload/3327/3327fig5.jpg" alt="Figure 5" /> 
Figure 5. Experimental approach. Custom-oligo nucleotides for dgn and dgnFS are cloned into the pEGFP-C1 vector and viruses are produced for each construct using HEK 293FT cells. The titer of the viruses is determined. Cells are transduced with the virus and expanded. After the favored treatment the cells are analyzed for fluorescence signal by flow cytometry.
<img src="/files/ftp_upload/3327/3327fig6.jpg" alt="Figure 6" /> 
Figure 6. Map of pLenti GFP-dgn. The map of the pLenti6/V5-DEST vector including the GFP-dgn sequence is displayed. Abbreviations: PCMV (CMV promoter), GFP-dgn (sequence of GFP-dgn), PSV40 (SV40 early promoter), EM7 (EM7 promoter), Blasticidin (Blasticidin resistance gene), &Delta;U3/3'LTR (3'LTR with deleted U3 region), SV40 pA (SV40 polyadenylation signal), Ampicillin (Ampicillin resistance gene), pUC ori (pUC origin), PRSV/5'LTR (RSV/5'LTR hybrid promoter), &Psi; (HIV-1 &Psi; packaging signal), RRE (HIV-1 Rev response element).
<img src="/files/ftp_upload/3327/3327fig7.jpg" alt="Figure 7" /> 
Figure 7. U2-OS titering plate. U2-OS were seeded out on a 6-well plate and transfected with different virus concentrations (dilution of 1/100 and 1/10, 1, 5 and 10 &mu;l of concentrated viral supernatant). One well was used as untransfected control (NT). After 6-7 days, the cells were stained with crystal violet and air dried.

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No conflicts of interest declared.

The first publication using green fluorescent protein (GFP) as a reporter substrate for ubiquitin-proteasome activity was published in 2000 12. Since then, GFP has become a common tool to visualize cellular activities, especially the ubiquitin-proteasome process. To monitor ubiquitin-proteasome activity in vivo a transgenic mouse model with a GFP-based reporter has been introduced 13. Additional in vivo research established another transgenic mouse model with a similar degron-desta...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>pEGFP-C1 Vector</td>
 <td>BD Bioscience Clontech</td>
 <td>6084-1</td>
 </tr>
 <tr>
 <td>pENTR Directional TOPO Cloning Kit</td>
 <td>Invitrogen</td>
 <td>K2400-20</td>
 </tr>
 <tr>
 <td>pLenti6/V5 Directional TOPO Cloning Kit</td>
 <td>Invitrogen</td>
 <td>V496-10</td>
 </tr>
 <tr>
 <td>Lipofectamine 2000 Reagent</td>
 <td>Invitrogen</td>
 <td>11668019</td>
 </tr>
 <tr>
 <td>DMEM</td>
 <td>Sigma</td>
 <td>D5546</td>
 </tr>
 <tr>
 <td>PVDF filter 			(Rotilabo-Spritzenfilter) </td>
 <td>Roth</td>
 <td>P667.1</td>
 </tr>
 <tr>
 <td>Polyethylene glycol</td>
 <td>Sigma</td>
 <td>P2139</td>
 </tr>
 <tr>
 <td>NaCl</td>
 <td>Merck</td>
 <td>1.06404.1000</td>
 </tr>
 <tr>
 <td>Dulbecco's Phosphate Buffered Saline 1x (PBS)</td>
 <td>Invitrogen</td>
 <td>14190</td>
 </tr>
 <tr>
 <td>hexadimethrine bromide</td>
 <td>Sigma</td>
 <td>10,768-9</td>
 </tr>
 <tr>
 <td>Blasticidin</td>
 <td>Invitrogen</td>
 <td>R21001</td>
 </tr>
 <tr>
 <td>Crystal violet</td>
 <td>Sigma</td>
 <td>C3886</td>
 </tr>
 <tr>
 <td>FACS tubes</td>
 <td>BD Biosciences</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Penicillin Streptomycin (Pen-Strep)</td>
 <td>Invitrogen</td>
 <td>15140130</td>
 </tr>
 <tr>
 <td>L-glutamine 200 mM</td>
 <td>Invitrogen</td>
 <td>25030024</td>
 </tr>
 <tr>
 <td>Fetal Bovine Serum (FBS)</td>
 <td>Biochrom AG</td>
 <td>S0115</td>
 </tr>
 <tr>
 <td>MEM Non-Essential Amino Acids (NEAA) 100x</td>
 <td>Invitrogen</td>
 <td>11140035</td>
 </tr>
 <tr>
 <td>MEM Sodium Pyruvate 100 mM</td>
 <td>Invitrogen</td>
 <td>11360039</td>
 </tr>
 <tr>
 <td>D-(+)-Glucose (45%)</td>
 <td>Sigma</td>
 <td>G8769</td>
 </tr>
 <tr>
 <td>Geneticin</td>
 <td>Invitrogen</td>
 <td>11811023</td>
 </tr>
 <tr>
 <td>CaCl2</td>
 <td>Merck</td>
 <td>C5080</td>
 </tr>
 <tr>
 <td>Hepes</td>
 <td>Sigma</td>
 <td>H3375</td>
 </tr>
 <tr>
 <td>Trypsin-EDTA (0.05%)</td>
 <td>Invitrogen</td>
 <td>25300054</td>
 </tr>
 </tbody>
</table>

monitoring of ubiquitin-proteasome activity in living cells using a degron (dgn)-destabilized green fluorescent protein (gfp)-based reporter protein

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. Maintenance of proteasome activity was implicated in many key cellular processes, like cell's stress response 3, cell cycle regulation and cellular differentiation 4 or in immune system response 5. The dysfunction of the ubiquitin-proteasome system has been related to the development of tumors and neurodegenerative diseases 4, 6. Additionally, a decrease in proteasome activity was found as a feature of cellular senescence and organismal aging 7, 8, 9, 10. Here, we present a method to measure ubiquitin-proteasome activity in living cells using a GFP-dgn fusion protein. To be able to monitor ubiquitin-proteasome activity in living primary cells, complementary DNA constructs coding for a green fluorescent protein (GFP)&#x2013;dgn fusion protein (GFP&#x2013;dgn, unstable) and a variant carrying a frameshift mutation (GFP&#x2013;dgnFS, stable 11) are inserted in lentiviral expression vectors. We prefer this technique over traditional transfection techniques because it guarantees a very high transfection efficiency independent of the cell type or the age of the donor. The difference between fluorescence displayed by the GFP&#x2013;dgnFS (stable) protein and the destabilized protein (GFP-dgn) in the absence or presence of proteasome inhibitor can be used to estimate ubiquitin-proteasome activity in each particular cell strain. These differences can be monitored by epifluorescence microscopy or can be measured by flow cytometry.

Watch this Scientific Journal Video about Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein at JoVE.com

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein

A method to monitor ubiquitin-proteasome activity in living cells is described. A degron-destabilized GFP- (GFP-dgn) and a stable GFP-dgnFS fusion protein are generated and transduced into the cell using a lentiviral expression vector. This technique allows to generate a stable GFP-dgn/GFP-dgnFS expressing cell line in which ubiquitin-proteasome activity can be easily assessed using epifluorescence or flow cytometry.

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. ...

monitoring-ubiquitin-proteasome-activity-living-cells-using-degron

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16.6K Views.  Institute for Biomedical Aging Research. A method to monitor ubiquitin-proteasome activity in living cells is described. A degron-destabilized GFP- (GFP-dgn) and a stable GFP-dgnFS fusion protein are generated and transduced into the cell using a lentiviral expression vector. This technique allows to generate a stable GFP-dgn/GFP-dgnFS expressing cell line in which ubiquitin-proteasome activity can be easily assessed using epifluorescence or flow cytometry.

Video: Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

Photobleaching Assays (FRAP &amp; FLIP) to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with good spatial and temporal resolution. Here we describe how to perform FRAP and FLIP assays of chromatin proteins, including H1 and HP1, in mouse embryonic stem (ES) cells. In a FRAP experiment, cells are transfected, either transiently or stably, with a protein of interest fused with the green fluorescent protein (GFP) or derivatives thereof (YFP, CFP, Cherry, etc.). In the transfected, fluorescing cells, an intense focused laser beam bleaches a relatively small region of interest (ROI). The laser wavelength is selected according to the fluorescent protein used for fusion. The laser light irreversibly bleaches the fluorescent signal of molecules in the ROI and, immediately following bleaching, the recovery of the fluorescent signal in the bleached area - mediated by the replacement of the bleached molecules with the unbleached molecules - is monitored using time lapse imaging. The generated fluorescence recovery curves provide information on the protein's mobility. If the fluorescent molecules are immobile, no fluorescence recovery will be observed. In a complementary approach, Fluorescence Loss in Photobleaching (FLIP), the laser beam bleaches the same spot repeatedly and the signal intensity is measured elsewhere in the fluorescing cell. FLIP experiments therefore measure signal decay rather than fluorescence recovery and are useful to determine protein mobility as well as protein shuttling between cellular compartments. Transient binding is a common property of chromatin-associated proteins. Although the major fraction of each chromatin protein is bound to chromatin at any given moment at steady state, the binding is transient and most chromatin proteins have a high turnover on chromatin, with a residence time in the order of seconds. These properties are crucial for generating high plasticity in genome expression1. Photobleaching experiments are therefore particularly useful to determine chromatin plasticity using GFP-fusion versions of chromatin structural proteins, especially in ES cells, where the dynamic exchange of chromatin proteins (including heterochromatin protein 1 (HP1), linker histone H1 and core histones) is higher than in differentiated cells2,3.

Photobleaching Assays FRAP &amp; FLIP to Measure Chromatin Protein Dynamics in Living Embryonic Stem Cells

We describe photobleaching methods including Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) to monitor chromatin protein dynamics in embryonic stem (ES) cells. Chromatin protein dynamics, which is considered to be one of the means to study chromatin plasticity, is enhanced in pluripotent cells.

Fluorescence Recovery After Photobleaching (FRAP) and Fluorescence Loss In Photobleaching (FLIP) enable the study of protein dynamics in living cells with ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.