The work in our laboratory is supported by grants from NIH, USDA National Institute of Food and Agriculture, NSF, BARD, DOE, and BSF to V.C.

1. Expression and extraction of the proteins 
<ol>
	<li>Differentially tag the proteins to be tested for their detection. Label the protein to be immobilized on the membrane (ProIM) with a tag of a larger size (e.g., GST), and the unfused tag can be used as an immobilized negative control (ProIMnc). Label the protein to be used as a soluble probe (ProSOL) with either a large or a small tag. Fuse the same tag to an appropriate negative control soluble protein (ProSOLnc) and include in the interaction assay to confirm the specificity of the detected binding.</li>
	<li>Choose the protein expression system to express tagged proteins, i.e., E. coli, baculovirus, etc., depending on the requirement for potential post translational modifications and yields of the proteins. </li>
	<li>Extract ProIM and ProIMnc from the organism of choice (step 1.2) using SDS-PAGE loading buffer (20% glycerol, 4% SDS, 20 mM Tris-HCl, pH6.8) containing proteinase inhibitor cocktail. Leave the cell suspension at room temperature for 15 min, add 0.5% of 2-mercaptoethanol and boil for 5 min.</li> 
	<li>Extract ProSOL and ProSOLnc from the organism of choice (step 1.2) using a standard protocols 16. These proteins should be readily soluble in the binding buffer (140 mM NaCl, 10 mM, Tris-HCl pH 7.4, 2 mM EDTA, 2 mM DTT, 1 % BSA, 0.1% Tween 20) used in the step 4.1. </li>
	<li>Determine the concentration of the recombinant proteins using a standard method, such as Bio-Rad protein assay kit. such as Bio-Rad protein assay kit. If crude extract, rather than purified protein, is used for the assay, estimate the concentration of the protein of interest in this extract by scanning densitometry of the corresponding band on an SDS-polyacrylamide gel stained with Coomassie Brilliant Blue R-250 and using known concentrations of BSA as reference.</li>
</ol>
2. Immobilization of ProIM and ProIMnc on the membrane
<ol>
	<li>Resolve two sets of extracts, each containing 1 &mu;g of ProIM and ProIMnc on an SDS-polyacrylamide gel according to the standard protocol 16.</li> 
	<li>After electrophoresis, place the transfer the gel to in 100 ml of transfer buffer (60 mM Glycine, 10 mM Tris, 0.0006% SDS, 20% MeOH) and incubate with gentle agitation for 20 min at room temperature.</li> 
	<li>Electrotransfer the proteins contained in the gel to a nitrocellulose membrane according to the standard protocol 16.</li>
</ol>
3. Re-folding of membrane-bound proteins 
<ol>
	<li>After electrotransfer, incubate the membrane for 15 min in 15 ml of buffer A (30 mM Tris-HCl pH7.4, 0.05% Tween 20) with gentle agitation to remove residual SDS.</li> 
	<li>After draining carefully, transfer the membrane to 25 ml of denaturation buffer (7 M guanidine hydrochloride, 2 mM EDTA, 50 mM DTT, 50 mM Tris-HCl pH 8.3 ). and incubate for 2 hours at room temperature with gentle agitation. (Note: The nitrocellulose membrane becomes opaque when incubated in the denaturation buffer). and incubate for 2 hours at room temperature with gentle agitation.</li>
	<li>Transfer the membrane to 25 ml of TBS (10 mM Tris-HCl, 150 mM NaCl, pH 7.4) and incubate with gentle agitation for 5 min (Note: During this step, the membrane regains its original white color).</li>
	<li>Transfer the membrane to 25 ml of binding buffer (see step 1.2) and incubate at 4&deg;C with gentle agitation for overnight.</li>
</ol>
4. Probing the ProIM by ProSOL
<ol>
	<li>Cut the membrane into two strips, both containing ProIM and ProIMnc.</li>
	<li>Make the ProSOL and ProSOLnc hybridization solutions by diluting the preparation with 1-10 &mu;g of either ProSOL or ProSOLnc in 10 ml of fresh binding buffer. Transfer each membrane into the ProSOL or ProSOLnc hybridization solution and incubate for 1.5 h at room temperature with gentle agitation.</li>
	<li>Remove the membranes from the hybridization solutions and rinse three times for 15 min each in TBS.</li>
</ol>
5. Visualizing protein-protein interaction by immunoblotting
<ol>
<li>Block the membrane with 2.5% skim milk in TBST (10 mM Tris-HCl, 140 mM NaCl, 0.05% Tween 20, pH 7.4) for 1 h at room temperature. Dilute the primary antibody (anti-strepII polyclonal rabbit antibody) in 0.5% skim milk in TBST at the concentration recommended by the manufacturer. </li>
<li>Place the blocked membranes in the antibody solution, and incubated for 1 h at room temperature or for overnight at 4&deg;C with gentle agitation.</li>
<li>Rinse the membranes in 20 ml TBST for 15 min, and twice for 5 min at room temperature with gentle agitation.</li>
<li>Dilute the secondary antibody (anti-rabbit IgG antibody) conjugated with horse radish peroxidase (HRP) in 0.5% skim milk in TBST at the concentration recommended by the manufacturer. Place the membranes in the secondary antibody solution and incubate for 1 h at room temperature with gentle agitation.</li>
<li>Rinse the membranes in 20 ml TBST for 15 min, and twice for 5 min at room temperature with gentle agitation. After the final rinse in TBS, visualize the protein-protein interaction using a HRP chemiluminescence substrate (for example, Millipore Immobilon western chemiluminescent HRP substrate).</li>
<li>Probe the same membranes with the primary antibody for the tag fused to ProIM, followed by the appropriate secondary antibody as described in steps 5.1 to 5.4. Visualize ProIM and ProIMnc as described in step 5.5 to validate the identity of the bands obtained in the protein membrane overlay assay (step 5.5). In most cases, stripping is not necessary, because the signal obtained by this step is much stronger than residual signal obtained from step 5.5.</li>
</ol>
6. Representative Results: 
 The ANK-MP interaction was observed by BiFC in tobacco epidermal cells (Figure 1A). Because MP is a highly insoluble protein when expressed in bacteria or in plants, the protein membrane overlay assay was adopted to validate this interaction in vitro (Figure 1B). Protein extracts containing 1 &mu;g of GST-MP (ProIM) or unfused GST (ProIMnc) were resolved by SDS-polyacrylamide gel electrophoresis, followed by electrotransfer to a nitrocellulose membrane. When these ProIMs were probed with soluble ANK-strepII (ProSOL), GST-MP, but not unfused GST, exhibited binding (Figure 1B, lanes 1, 2, compare to lanes 5, 6). Moreover, when the same set of ProIMs were probed with an unrelated ProSOLnc, i.e., Arabidopsis cytoplasmic NADH kinase tagged strepII (NADH3-strepII), no binding was not observed, further demonstrating the specificity of the ANK-MP interaction (Figure 1B, lanes 3, 4). 
<img src="/files/ftp_upload/2961/2961fig1.jpg" alt="Figure 1"/> 
Figure 1. Specific binding of tobacco ANK to TMV MP in vivo and in vitro. (A) ANK-MP interaction in living tobacco epidermal cells as detected by BiFC. Strong YFP signal was reconstructed when MP and ANK, fused to C-terminal and N-terminal halves of YFP, respectively, were coexpressed in tobacco epidermis following microbombardment of their encoding genes. This BiFC signal accumulated in puncta at the cell periphery, which are diagnostic of plasmodesmata 15. (B) ANK-MP interaction in vitro as detected by protein membrane overlay assay. Protein extracts containing 1 &mu;g of GST-MP (ProIM) or unfused GST (ProIMnc) were resolved on a 15% SDS-polyacrylamide gel, followed by electrotransfer onto a nitrocellulose membrane. The GST-MPProIM and GSTProIMnc were incubated with 0.5 &mu;g/ml of ANK-strepII (ProSOL), and ANK binding was detected by probing the membrane with anti-strepII rabbit polyclonal antibody, followed by anti-rabbit IgG+M secondary antibody conjugated to HRP (lanes 1 and 2). Neither GST-MPProIM nor GSTProIMnc interacted with an unrelated protein, Arabidopsis cytoplasmic NADH kinase, fused to the strepII tag (ProSOLnc, lanes 3 and 4). The identity of the band observed in this assay was confirmed by probing the membrane with anti-GST antibody (lanes 5 and 6). When the membrane was treated with denaturation buffer without being washed with buffer A, the binding of the GST-MP to ANK is lost, while unidentified proteins contained in the GST-MP and GST contining protein extracts reacted with ANK-strepII, demonstrating the importance of the step 3.1 before the denaturation process (lanes 7 and 8).

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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+implementation+of+bimolecular+fluorescence+complementation+(BiFC)+assays+for+the+visualization+of+protein+interactions+in+living+cells.">Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells.</a> Nat Protoc. 1, 1278-1286 (2006).</li><li>Shyu, Y. 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T., Taylor, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> , (2011).</li><li>Tzfira, T., Vaidya, M., Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=VIP1,+an+Arabidopsis+protein+that+interacts+with+Agrobacterium+VirE2,+is+involved+in+VirE2+nuclear+import+and+Agrobacterium+infectivity.">VIP1, an Arabidopsis protein that interacts with Agrobacterium VirE2, is involved in VirE2 nuclear import and Agrobacterium infectivity.</a> EMBO J. 20, 3596-3607 (2001).</li><li>Chen, M. 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No conflicts of interest declared.

This approach is suitable for testing protein-protein interactions between combinations of the proteins,when at least one of which the proteins is readily soluble in the binding buffer, and was successfully applied to other combination of proteins 17,18. The iInteractions between the proteins that are both insoluble under these conditions cannot be tested by this protocol. 
Also, successful refolding of ProIM is critical for the assay. Rinsing the membrane in TBS after the electro...

<table border="1">
<tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalog number</td>
 </tr>
 <tr>
 <td>Protein assay kit</td>
 <td>Bio-Rad</td>
 <td>500-0001</td>
 </tr>
 <tr>
 <td>Proteinase inhibitor cocktail </td>
 <td>SIGMA </td>
 <td>S8820 </td>
 </tr>
 <tr>
 <td>Mini-PROTEAN system </td>
 <td>Bio-RAD </td>
 <td>165-8000 </td>
 </tr>
 <tr>
 <td>Semi-dry western blotting SD electrotransfer system </td>
 <td>Bio-RAD </td>
 <td>170-3940 </td>
 </tr>
 <tr>
 <td>Anti-rabbit IgG antibody conjugated with horse radish peroxidase </td>
 <td>GenScript </td>
 <td>A00098 </td>
 </tr>
 <tr>
 <td>Anti-GST rabbit polyclonal antibody </td>
 <td>GenScript </td>
 <td>A00097 </td>
 </tr>
 <tr>
 <td>Anti-strepII </td>
 <td>GenScript </td>
 <td>A00626 </td>
 </tr>
 <tr>
 <td>BioTrace, NT nitrocellulose transfer membrane </td>
 <td>Pall Scientific </td>
 <td>27377-000 </td>
 </tr>
 <tr>
 <td>Immobilon western chemiluminescent HRP substrate </td>
 <td>Millipore </td>
 <td>WBKL S0 050 </td>
 </tr>
 </tbody>
</table>

protein membrane overlay assay: a protocol to test interaction between soluble and insoluble proteins in vitro

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level. There are several methods, both in vitro and in vivo, to evaluate protein binding, and at least two methods that complement the shortcomings of each other should be conducted to obtain reliable insights. 
For an in vivo assay, the bimolecular fluorescence complementation (BiFC) assay represents the most popular and least invasive approach that enables to detect protein-protein interaction within living cells, as well as identify the intracellular localization of the interacting proteins 1,2. In this assay, non-fluorescent N- and C-terminal halves of GFP or its variants are fused to tested proteins, and when the two fusion proteins are brought together due to the tested proteins&#x2019; interactions, the fluorescent signal is reconstituted3-6. Because its signal is readily detectable by epifluorescence or confocal microscopy, BiFC has emerged as a powerful tool of choice among cell biologists for studying about protein-protein interactions in living cells 3. This assay, however, can sometimes produce false positive results. For example, the fluorescent signal can be reconstituted by two GFP fragments arranged as far as 7 nm from each other due to close packing in a small subcellular compartment, rather that due to specific interactions7.
Due to these limitations, the results obtained from live cell imaging technologies should be confirmed by an independent approach based on a different principle for detecting protein interactions. Co-immunoprecipitation (Co-IP) or glutathione transferase (GST) pull-down assays represent such alternative methods that are commonly used to analyze protein-protein interactions in vitro. However, iIn these assays, however, the tested proteins must be readily soluble in the buffer that supportsused for the binding reaction. Therefore, specific interactions involving an insoluble protein cannot be assessed by these techniques.
 Here, we illustrate the protocol for the protein membrane overlay binding assay, which circumvents this difficulty. In this technique, interaction between soluble and insoluble proteins can be reliably tested because one of the proteins is immobilized on a membrane matrix. This method, in combination with in vivo experiments, such as BiFC, provides a reliable approach to investigate and characterize interactions faithfully between soluble and insoluble proteins. In this article, binding between Tobacco mosaic virus (TMV) movement protein (MP), which exerts multiple functions during viral cell-to-cell transport8-14, and a recently identified plant cellular interactor, tobacco ankyrin repeat-containing protein (ANK) 15, is demonstrated using this technique.

Watch this Scientific Journal Video about Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro at JoVE.com

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Testing protein-protein interaction is indispensable for dissection of protein functionality. Here, we introduce an in vitro protein-protein binding assay to probe a membrane-immobilized protein with a soluble protein. This assay provides a reliable method to test interaction between an insoluble protein and a protein in solution.

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

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22.0K Aufrufe.  State University of New York. Testen Protein-Protein-Interaktion ist für die Präparation von Protein-Funktionalität unverzichtbar. Hier stellen wir eine In-vitro- Protein-Protein-Bindungs-Assay auf eine Membran-immobilisierten Protein mit ein lösliches Protein-Sonde. Dieser Test liefert eine zuverlässige Methode, um die Interaktion zwischen einem unlöslichen Protein und ein Protein in Lösung zu testen.

Serie: Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

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 ...

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

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 ...

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 ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&amp;FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

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 ...

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel protein-protein interaction pairs and monitoring protein interaction dynamics are of particular interest for revealing how plants respond to environmental factors and/or developmental signals. A plethora of approaches have been developed to examine protein-protein interactions, either in vitro or in vivo. Among them, the recently established luciferase complementation imaging (LCI) assay is the simplest and fastest method for demonstrating in vivo protein-protein interactions. In this assay, protein A or protein B is fused with the amino-terminal or carboxyl-terminal half of luciferase, respectively. When protein A interacts with protein B, the two halves of luciferase will be reconstituted to form a functional and active luciferase enzyme. Luciferase activity can be recorded with a luminometer or CCD-camera. Compared with other approaches, the LCI assay shows protein-protein interactions both qualitatively and quantitatively. Agrobacterium infiltration in Nicotiana benthamiana leaves is a widely used system for transient protein expression. With the combination of LCI and transient expression, these approaches show that the physical interaction between COP1 and SPA1 was gradually reduced after jasmonate treatment.

Luciferase Complementation Imaging Assay in Nicotiana benthamiana Leaves for Transiently Determining Protein-protein Interaction Dynamics

This manuscript describes an easy and rapid experimental procedure for determining protein-protein interactions based on the measurement of luciferase activity.

Protein-protein interactions are fundamental mechanisms for relaying signal transduction in most cellular processes; therefore, identification of novel ...