We thank Drs. Steve Mount and Xiaoning Zhang for the 35S::GFP pGlowbug construct and the Hokensen Doctoral Fellowship to C. H. Research in Z. L's laboratory is supported by the US National Science Foundation (IOB0616096 and MCB0744752). Z.L. is partially supported by the University of Maryland Agricultural Experiment Station.

I. Plasmid preparation
The plasmid DNA used for bombardment must be of high purity and at a concentration of 500ng/&mu;l or greater. For BiFC involving two plasmid constructs, 25 &mu;g of each plasmid is needed. In other words, 1:1 molar ratio of the two plasmids should be mixed to give rise to 50 &mu;g total plasmid DNA for the preparation of cartridges. Be mindful that adding more DNA than 50 &mu;g may cause agglomeration of the gold particles and should be avoided. Commercially available plasmid DNA extraction kits are recommended for isolating plasmid DNAs.
Plasmid vectors should be relatively small in size such as pUC19. We cloned SEU and LUH cDNA into pUC19-SPYNE and pUC19-SPYCE, respectively3. Binary vectors for Agrobacterium-mediated transformation are too large and inappropriate for the bombardment.
II. Cartridge preparation
Cartridge preparation involves coating gold particles with plasmid DNA and loading them into plastic tubing that is subsequently cut into half inch long pieces, which can be loaded into the gene gun cartridge holders. Cartridge preparation is described in great details in a separate Jove article4 and is thus not described here. For bombardment of onion cells, we recommend mixing 50 &mu;g of plasmid DNA with 12.5 mg of gold particles per preparation, which yields about up to 50 cartridges at 1 &mu;g DNA/0.25 mg gold/cartridge. Each cartridge is for one shot. The gold particles can be either 0.6 &mu;m or 1.0 &mu;m in diameter (Cat #1652262 or Cat #1652263; Biorad). We also recommend using 0.5 mg/ml PVP solution for the cartridge preparation. For BiFC, combine the two plasmid DNAs of each putative interacting partner in the same cartridge preparation.
After cartridge preparation, it is important to test the cartridges by firing the newly made cartridges at a helium pressure between 150 to 200 psi into a square Parafilm that is wrapped around a Petri dish. This will test if the gold particles can be propelled efficiently from the cartridges; you should see faint gold particles on the Parafilm. This gives you an idea of the area that each shot will cover. You may notice different amounts of gold particles propelled onto the Parafilm at different helium pressures (at 150-200 psi range). If there is no significant difference, choose the lower pressure for bombardment.
III. Preparing onion tissues
On the day of shooting, peel off outer skin from a large yellow onion. Using a clean and sharp razor blade, cut 4-5 layers deep a rectangular area of about 2 X 8 cm. Take out the rectangular onion tissue, discard the outer two layers, cut the remaining layers of onion into about 2X1.5 cm pieces. Place the onion pieces in a Petri dish containing filter paper (Whatman 3MM paper cut into circles) moistened with sterile water. Cover the Petri dish and they are ready to go.
IV. Bombardment
<ol>
 <li> Load the cartridges into the white round cartridge holders. Cartridges containing different plasmid constructs should be loaded into different cartridge holders. Each cartridge holder can hold up to 12 cartridges for 12 shots.</li>
 <li> Insert the battery and the fresh barrel liner into the Helios Gene Gun. Make sure that both the barrel liner and the gun have respective o-rings attached.</li>
 <li> First, insert an empty cartridge holder into the gene gun, with mark #12 on the cartridge holder facing the top of the gun. Advance cartridge holder once or twice to make sure it is inserted correctly. </li>
 <li> Attach gun to the helium tank and adjust pressure between 150 to 200 psi.</li>
 <li> Wear ear protection and fire gun a few times with the empty cartridge holder to clean out the chamber of the gun and to make sure that the pressure is stable. </li>
 <li> Remove the empty cartridge holder and insert a loaded cartridge holder. Advance the cartridge holder so that the cartridge you want to fire is positioned at bottom (6 o'clock) of the cartridge holder. Shooting a piece of onion here provides a form of negative control for background fluorescence.</li>
 <li> Place onion pieces in the middle of an empty Petri dish with the inner most layer of the onion facing up. Rest the gene gun's barrel liner on Petri dish and fire the gun by holding down the side button while pulling the trigger. Transfer the bombarded onion to a Petri dish containing moist 3MM filter paper. </li>
 <li> Advance to the next cartridge and fire a different onion piece. Repeat this step until four to five pieces of onions are shot. Each onion piece is shot once.</li>
 <li> Change to a different cartridge holder loaded with cartridges containing a different plasmid or different plasmid combination. Remember to change the barrel liner as well to prevent contamination. Shoot four to five onion pieces. </li>
 <li> After bombardment, return all onions to perti dishes. Cover the Petri dishes with lids. Wrap them with Parafilm to prevent drying. Incubate the onion pieces at room temperature in the dark for 16-48 hours.</li>
 <li> Turn off gas and release pressure before detaching the gene gun from the helium tank. Unscrew the barrel liner, and remove the cartridge holder and the used (or unused) cartridges.</li>
 <li> Cleaning cartridge holders and barrel liners by submerging them in a beaker with soapy water. Place the beaker in a bath sonicator and sonicate for 20 minutes. Afterwards, rinse them with water to remove all soap residues. Soak them in 70% ethanol for an hour and then dry them on paper towels.</li>
</ol>
V. Observation
<ol>
 <li> After incubating bombarded onion pieces for 16-20 hours at room temperature, we can observe GFP or YFP fluorescence using a fluorescence microscope such as the Zeiss Axio Observer.z1 used in this study.</li>
 <li> Use forceps with flat ends to slowly peel a single cell layer off the inner epidermis of the onion (the layer that directly faces the gun during the bombardment). Place the peel onto a drop of water on a slide. Add coverslip but be careful to minimize bubbles.</li>
 <li> Under bright field of the microscope, first check for cytoplasmic streaming to ensure that the cells are alive. To do so, expose the slide to the bright field light for a few minutes to warm up the onion tissue and then look for moving plastids. Cytoplasmic streaming is not always easy to see, so don't get discouraged if you can't see any.</li>
 <li> For fluorescence detection, use appropriate excitation and detection filters for GFP or YFP. It is important to include negative controls such as onion pieces shot with a non-fluorescent plasmid. Faint yellow signals may appear from wounded cells, air bubbles, or debris. It is also important to include positive controls. A plasmid containing constitutively expressed fluorescent reporter such as 35S::GFP is an excellent positive control. Visible GFP signals from onion epidermal cells (Figure 1A, D) indicated that the cartridges, the onion tissues, and the gene gun firing are properly prepared and executed. Subsequently, we observed interactions between SEU-pSPYNE and LUH-pSPYCE in onion pieces bombarded with cartridges containing a mixture of 35S::SEU-pSPYNE and 35S::LUH-pSPYCE DNAs. Faint YFP fluorescence was observed in nucleus only (Figure 1 B, E), indicating the direct physical interaction between SEU and LUH that brought the split YFP fragments together in the nucleus. Note that the YFP signal from BiFC is significantly weaker than the GFP signal from 35S::GFP.</li>
</ol>
Representative Results
In our study reported here, the 35S::GFP positive control plasmid gives off strong fluorescence that fills the entire cell, including both the nucleus and cytoplasm (Figure 1A, D). GFP protein is small enough to diffuse into nucleus without a nuclear localization signal. In contrast, SEU and LUH are two Arabidopsis transcriptional factors previously shown to interact in a yeast two-hybrid assay2. SEU-pSPYNE (SEU fused to the N-terminal fragment of YFP) and LUH-pSPYCE (SEU fused to the N-terminal fragment of YFP), both too large to diffuse into the nucleus, contain nuclear localization signals2,5. The YFP fluorescent signal detected in the onion cell nucleus (Figure 1 B, E) indicates that SEU-pSPYNE and LUH-pSPYCE can interact in onion nuclei. No fluorescent signal is detected in onions bombarded with plasmid mix of vector plasmid pSPYNE containing the N-terminal fragment of YFP and LUH-pSPYCE (Figure 1C, F).
The BiFC-mediated YFP fluorescence is significantly weaker than the GFP-mediated fluorescence from the 35S::GFP plasmid. In our study, they also showed different subcellular localization (compare Figure 1A with B). Additionally, the number of fluorescent cells is significantly lower for BiFC, as BiFC only works when both partner plasmids successfully enter and are expressed in the same onion cells.

<img src="/files/ftp_upload/1963/1963fig1.jpg" alt="Figure 1" /> 
Figure 1. Fluorescent (A, B, C) and bright field (D, E, F) images of onion cells bombarded with different plasmid constructs. (A) and (D), Onion cells bombarded with 35S::GFP. GFP diffuses through out the cytoplasm and nucleus. (B) and (E), Onion cells bombarded with equal molar mixes of 35S::SEU-pSPYNE (SEU fused to the N-terminal fragment of YFP) and 35S::LUH-pSPYCE (LUH fused to the C-terminal fragment) plasmid DNA. An interaction between SEU-pSPYNE and LUH-pSPYCE is shown by a fluorescent nucleus. (C) and (F), A negative control showing onion cells bombarded with mixes of vector pUC19-SPYNE and LUH-pSPYCE plasmids. No fluorescent signal is detected.

<ol>
	<li>Hu, C. D., Chinenov, Y., Kerppola, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+interactions+among+bZIP+and+Rel+family+proteins+in+living+cells+using+bimolecular+fluorescence+complementation.">Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation.</a> Mol Cell. 9 (4), 789-789 (2002).</li><li>Sitaraman, J., Bui, M., Liu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LEUNIG_HOMOLOG+and+LEUNIG+perform+partially+redundant+functions+during+Arabidopsis+embryo+and+floral+development.">LEUNIG_HOMOLOG and LEUNIG perform partially redundant functions during Arabidopsis embryo and floral development.</a> Plant Physiol. 147 (2), 672-672 (2008).</li><li>Walter, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+protein+interactions+in+living+plant+cells+using+bimolecular+fluorescence+complementation.">Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation.</a> Plant J. 40 (3), 428-428 (2004).</li><li>Woods, G., Zito, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+gene+gun+bullets+and+biolistic+transfection+of+neurons+in+slice+culture.">Preparation of gene gun bullets and biolistic transfection of neurons in slice culture.</a> J Vis Exp. , (2008).</li><li>Azhakanandam, S., Nole-Wilson, S., Bao, F., Franks, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SEUSS+and+AINTEGUMENTA+mediate+patterning+and+ovule+initiation+during+gynoecium+medial+domain+development.">SEUSS and AINTEGUMENTA mediate patterning and ovule initiation during gynoecium medial domain development.</a> Plant Physiol. 146 (3), 1165-1165 (2008).</li><li>Zhang, X. N., Mount, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+alternatively+spliced+isoforms+of+the+Arabidopsis+SR45+protein+have+distinct+roles+during+normal+plant+development.">Two alternatively spliced isoforms of the Arabidopsis SR45 protein have distinct roles during normal plant development.</a> Plant Physiol. 150 (3), 1450-1450 (2009).</li></ol>

No conflicts of interest declared.

<ol>
<li>	For first time users, we recommend practicing the entire procedure, from cartridge preparation to observation, with a single plasmid constitutive reporter such as 35S::GFP or 35S::GUS. We used a 35S::GFP plasmid obtained from Drs. Steve Mount and Xiaoning Zhang6 (Figure 1A, D). The gene shot control cartridges sold at RioRad (Cat 165-2244) are not suitable for plants.</li>
<li>	For BiFC, a second positive control is necessary, which should consist of two known interacting partners fused to the C-terminal and N-terminal f...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Yellow onion</td>
<td>&nbsp;</td>
<td>Any supermarket</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Helios Gene Gun</td>
<td>&nbsp;</td>
<td>BioRad</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescent microscope with appropriate filters for GFP or YFP</td>
<td>&nbsp;</td>
<td>Zeiss, Nikon, Olympus</td>
<td>&nbsp;</td>
<td>&nbsp;</td>		</tbody>
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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.

Watch this Scientific Journal Video about Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun at JoVE.com

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)

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 ...

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19.5K Aufrufe.  University of Maryland. Dieser Artikel veranschaulicht, wie man richtig mit dem BioRad Helios Gene Gun, um Plasmid-DNA in Zwiebel Epidermiszellen einzuführen und wie man für Protein-Protein-Interaktionen in Zwiebel-Zellen auf dem Prinzip der bimolekularen Fluoreszenz Komplementation (BIFC) basierter Test

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

Gene-gun Transfection of Hippocampal Neurons

Preparation of Gene Gun Bullets and Biolistic Transfection of Neurons in Slice Culture

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed protocol for biolistic transfection of rat hippocampal slices, from the initial preparation of DNA coated bullets to the final shooting of the organotypic slice cultures using a gene gun. Gene gun transfection is an efficient and easy means of transfecting neurons and is especially useful for fluorescently labeling a small subset of cells in tissue slice. In this video, we first outline the steps required to coat gold particles with DNA. We next demonstrate how to line the inside of plastic tubing with the gold/DNA bullets, and how to cut this tubing to obtain the plastic cartridges for loading into the gene gun. Finally, we perform biolistic transfection of rat hippocampal slice cultures, demonstrating handling of the Bio-Rad Helios gene gun, and offering trouble shooting advice to obtain healthy and optimally transfected tissue slices.  

We describe a method for preparing DNA coated gold bullets and demonstrate the use of such bullets to biolistically transfect neurons in cultured hippocampal slices.

Biolistic transfection is a physical means of transfecting cells by bombarding tissue with high velocity DNA coated particles. We provide a detailed ...

Bimolecular Fluorescence Complementation

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as immunoprecipitation do not provide information on the spatial localization of complexes. In contrast, BiFC monitors the interaction and subcellular compartmentalization of protein complexes. In this method, a fluororescent protein is split into amino- and carboxy-terminal non-fluorescent fragments which are then fused to two proteins of interest. Interaction of the proteins results in reconstitution of the fluorophore (Figure 1)1,2. A limitation of BiFC is that once the fragmented fluorophore is reconstituted the complex is irreversible3. This limitation is advantageous in detecting transient or weak interactions, but precludes a kinetic analysis of complex dynamics. An additional caveat is that the reconstituted flourophore requires 30min to mature and fluoresce, again precluding the observation of real time interactions4. BiFC is a specific example of the protein fragment complementation assay (PCA) which employs reporter proteins such as green fluorescent protein variants (BiFC), dihydrofolate reductase, b-lactamase, and luciferase to measure protein:protein interactions5,6. Alternative methods to study protein:protein interactions in cells include fluorescence co-localization and F&ouml;rster resonance energy transfer (FRET)7. For co-localization, two proteins are individually tagged either directly with a fluorophore or by indirect immunofluorescence. However, this approach leads to high background of non-interacting proteins making it difficult to interpret co-localization data. In addition, due to the limits of resolution of confocal microscopy, two proteins may appear co-localized without necessarily interacting. With BiFC, fluorescence is only observed when the two proteins of interest interact. FRET is another excellent method for studying protein:protein interactions, but can be technically challenging. FRET experiments require the donor and acceptor to be of similar brightness and stoichiometry in the cell. In addition, one must account for bleed through of the donor into the acceptor channel and vice versa. Unlike FRET, BiFC has little background fluorescence, little post processing of image data, does not require high overexpression, and can detect weak or transient interactions. Bioluminescence resonance energy transfer (BRET) is a method similar to FRET except the donor is an enzyme (e.g. luciferase) that catalyzes a substrate to become bioluminescent thereby exciting an acceptor. BRET lacks the technical problems of bleed through and high background fluorescence but lacks the ability to provide spatial information due to the lack of substrate localization to specific compartments8. Overall, BiFC is an excellent method for visualizing subcellular localization of protein complexes to gain insight into compartmentalized signaling.

The subcellular localization of proteins is important in determining the spatio-temporal regulation of cell signaling. Here, we describe bimolecular fluorescence complementation (BiFC) as a straightforward method for monitoring the spatial interactions of proteins in the cell.

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as ...

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact 1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays10. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.
BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction.

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

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 ...

Fluorescence Imaging with One-nanometer Accuracy (FIONA)

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in the x-y plane. Here a summary of the FIONA technique is reported and examples of research that have been performed using FIONA are briefly described. First, how to set up the required equipment for FIONA experiments, i.e., a total internal reflection fluorescence microscopy (TIRFM), with details on aligning the optics, is described. Then how to carry out a simple FIONA experiment on localizing immobilized Cy3-DNA single molecules using appropriate protocols, followed by the use of FIONA to measure the 36 nm step size of a single truncated myosin Va motor labeled with a quantum dot, is illustrated. Lastly, recent effort to extend the application of FIONA to thick samples is reported. It is shown that, using a water immersion objective and quantum dots soaked deep in sol-gels and rabbit eye corneas (&#62;200 &#181;m), localization precision of 2-3 nm can be achieved.

Fluorescence Imaging with One-nanometer Accuracy FIONA

Single fluorophores can be localized with nanometer precision using FIONA. Here a summary of the FIONA technique is reported, and how to carry out FIONA experiments is described.

Fluorescence imaging with one-nanometer accuracy (FIONA) is a simple but useful technique for localizing single fluorophores with nanometer precision in ...