Name: dissecting multi-protein signaling complexes by bimolecular complementation affinity purification (bicap)
Uploaded: 2018-06-15T13:30:00
Description: Watch this Scientific Journal Video about Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP at JoVE.com

D.R.C is a Cancer Institute NSW Fellow and D.N.S was previously a Cancer Institute NSW Fellow. The research findings presented in this manuscript were funded by the Cancer Institute NSW (13/FRL/1-02 and 09/CDF/2-39), NHMRC (Project Grant GNT1052963), Science Foundation Ireland (11/SIRG/B2157), NSW Office of Science and Medical Research, Guest Family Fellowship and Mostyn Family Foundation. J.F.H. and R.S. were recipients of an Australian Postgraduate Award.

1. Plasmid Cloning
NOTE: To generate plasmid vectors with the V1 or V2 tags fused to either the N-terminus or C-terminus of the gene of interest, BiFC destination vectors have been deposited with Addgene [N-terminal tag: pDEST-V1-ORF (#73635), pDEST-V2-ORF (#73636). C-terminal tag: pDEST-ORF-V1 (#73637), pDEST-ORF-V2 (#73638)]. The gene/s of interest will need to be in specific recombination cloning compatible entry vectors (i.e., pDONR223 or pENTR221), without stop codons, to proceed w...

<ol>
	<li>Kolch, W., Pitt, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+proteomics+to+dissect+tyrosine+kinase+signalling+pathways+in+cancer.">Functional proteomics to dissect tyrosine kinase signalling pathways in cancer.</a> Nat Rev Cancer. 10 (9), 618-629 (2010).</li><li>Pawson, T., Kofler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinome+signaling+through+regulated+protein-protein+interactions+in+normal+and+cancer+cells.">Kinome signaling through regulated protein-protein interactions in normal and cancer cells.</a> Curr Opin Cell Biol. 21 (2), 147-153 (2009).</li><li>Croucher, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bimolecular+complementation+affinity+purification+(BiCAP)+reveals+dimer-specific+protein+interactions+for+ERBB2+dimers.">Bimolecular complementation affinity purification (BiCAP) reveals dimer-specific protein interactions for ERBB2 dimers.</a> Sci Signal. 9 (436), ra69 (2016).</li><li>Cassonnet, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Benchmarking+a+luciferase+complementation+assay+for+detecting+protein+complexes.">Benchmarking a luciferase complementation assay for detecting protein complexes.</a> Nat Methods. 8 (12), 990-992 (2011).</li><li>Rossi, F., Charlton, C. A., Blau, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+protein-protein+interactions+in+intact+eukaryotic+cells+by+beta-galactosidase+complementation.">Monitoring protein-protein interactions in intact eukaryotic cells by beta-galactosidase complementation.</a> Proc Natl Acad Sci U S A. 94 (16), 8405-8410 (1997).</li><li>Magliery, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+protein-protein+interactions+with+a+green+fluorescent+protein+fragment+reassembly+trap:+scope+and+mechanism.">Detecting protein-protein interactions with a green fluorescent protein fragment reassembly trap: scope and mechanism.</a> J Am Chem Soc. 127 (1), 146-157 (2005).</li><li>Hu, C. D., 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=Simultaneous+visualization+of+multiple+protein+interactions+in+living+cells+using+multicolor+fluorescence+complementation+analysis.">Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis.</a> Nat Biotechnol. 21 (5), 539-545 (2003).</li><li>Kubala, M. H., Kovtun, O., Alexandrov, K., Collins, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+and+thermodynamic+analysis+of+the+GFP:GFP-nanobody+complex.">Structural and thermodynamic analysis of the GFP:GFP-nanobody complex.</a> Protein Sci. 19 (12), 2389-2401 (2010).</li><li>Fegan, A., White, B., Carlson, J. C., Wagner, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemically+controlled+protein+assembly:+techniques+and+applications.">Chemically controlled protein assembly: techniques and applications.</a> Chem Rev. 110 (6), 3315-3336 (2010).</li><li>JoVE Science Education Database. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+methods+in+cellular+and+molecular+biology.+plasmid+purification.">Basic methods in cellular and molecular biology. plasmid purification.</a> J Vis Exp. , (2017).</li><li>Eslami, A., Lujan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Western+blotting:+sample+preparation+to+detection.">Western blotting: sample preparation to detection.</a> J Vis Exp. (44), (2010).</li><li>Shearer, R. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+E3+ubiquitin+ligase+UBR5+regulates+centriolar+satellite+stability+and+primary+cilia+formation+via+ubiquitylation+of+CSPP-L.">The E3 ubiquitin ligase UBR5 regulates centriolar satellite stability and primary cilia formation via ubiquitylation of CSPP-L.</a> Mol Biol Cell. , (2018).</li><li>Shannon, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoscape:+A+software+environment+for+integrated+models+of+biomolecular+interaction+networks.">Cytoscape: A software environment for integrated models of biomolecular interaction networks.</a> Genome Res. 13 (11), 2498-2504 (2003).</li><li>Schopp, I. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Split-BioID+a+conditional+proteomics+approach+to+monitor+the+composition+of+spatiotemporally+defined+protein+complexes.">Split-BioID a conditional proteomics approach to monitor the composition of spatiotemporally defined protein complexes.</a> Nat Commun. 8, 15690 (2017).</li></ol>

BiCAP is a powerful method for isolating specific protein dimers while excluding the individual components and their competing binding partners3. BiCAP is based on adaptation of a fluorescence protein complementation assay called BiFC6. Existing methods, including BiFC and proximity ligation assays, have been used extensively to visualize and quantify protein interactions in live cells7, but did not provide an effective means of isolating and charact...

Protein complex assembly is a key process in maintaining the spatiotemporal specificity of many signalling pathways1,2. While the critical nature of this regulatory role is widely recognized, there is a lack of experimental techniques available to scrutinize these complexes. Most interactomics studies focus upon interactions with individual proteins, or the sequential enrichment of individual complex components. Here we present a technique for the isolation of a specific protein dimer while excluding the individual moieties of the component proteins as well as complexes formed with competing binding partners3. We have called this technique Bimolecular Complementation Affinity Purification (BiCAP), as it is a combination of a previously existing protein fragment complementation assay, Bimolecular Fluorescence Complementation (BiFC), with the novel use of a conformation-specific recombinant nanobody towards GFP and its derivatives (see Table of Materials).
A typical protein-fragment complementation assay relies on the expression of &#34;bait&#34; and &#34;prey&#34; proteins fused to split fragments of reporters such as luciferase4, &#946;-galactosidase5, or green fluorescent protein (GFP)6 (Figure 1A). Through the interaction of the bait and prey proteins, the split reporter domains are encouraged to refold into a functional structure, allowing the interaction of the bait and prey proteins to be visualized or quantified. BiCAP was adapted from a version of this technique that made use of fragments of the GFP variant Venus. Fluorescent protein complementation assays are a popular method for visualizing protein-protein interactions in a live cell, but until now have been limited to this one function7. BiCAP represents a significant advance in this regard, as this technique not only allows for visualization, but also the isolation and interrogation of the resulting protein-protein interaction.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57109/57109fig1.jpg" /> 
Figure 1: The structural principal behind the BiCAP technique. (A) A schematic outlining the principal behind bimolecular fluorescence complementation showing the &#39;bait&#39; and &#39;prey&#39; proteins tagged with the N-terminal V1 or C-terminal V2 fragments of the full-length Venus protein. (B) Structural analysis of the interaction interface (cyan) between the GFP nanobody (green) and recombined Venus, showing the position of the V1 (grey) and V2 (orange) fragments (PDB accession 3OGO). This figure is republished fromCroucher et al.3 Reprinted with permission from AAAS. <a href="//cloudflare.jove.com/files/ftp_upload/57109/57109fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The BiCAP technique makes use of two non-fluorescent fragments of Venus (named V1 and V2) which associate with a low degree of affinity unless an interaction occurs between their fusion partners. In this instance, the two split domains refold into the functional &#946;-barrel structure of the fluorophore (Figure 1B)6. The key innovation of BiCAP comes from the introduction of the recombinant GFP nanobody, which recognizes a three-dimensional epitope on the &#946;-barrel of GFP (and variants such as Venus) which is only present on the correctly recombined and folded fluorophore (Figure 1B)8. Crucially, the GFP nanobody does not bind to either of the individual Venus fragments. This facilitates the isolation of protein dimers only after the two proteins have formed a complex of their own volition, leading to more representative results than those acquired from methods that make use of chemically-induced, forced interactions9.
BiCAP is a powerful technique that specifically focuses on multi-protein complexes, which can potentially be combined with a number of downstream applications to improve the granularity of our understanding of the role these complexes play in signal transduction. It also encompasses the important feature of allowing visualization of protein interactions in situ. To date, BiCAP has been demonstrated as an effective method of analyzing the interactome of receptor tyrosine kinase (RTK) dimers3, but the adaptability of this method means that it can be adopted in almost any protein interaction context.

<table>
	<tbody>
		<tr>
			<td>LR Clonase II Plus enzyme</td>
			<td>Thermo Fisher Scientific</td>
			<td>12538120</td>
			<td>Recombinase enzyme required for Gateway cloning (Step 1) into pDEST BiFC destination vectors</td>
		</tr>
		<tr>
			<td>Proteinase K, recombinant, PCR grade</td>
			<td>Thermo Fisher Scientific</td>
			<td>EO0491</td>
			<td></td>
		</tr>
		<tr>
			<td>14 mL round-bottomed polypropylene tube</td>
			<td>Corning</td>
			<td>352059</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Roche Diagnostics Australia</td>
			<td>10835242001</td>
			<td>Stock solution prepared at 100 &#956;g/mL in distilled water.</td>
		</tr>
		<tr>
			<td>Miniprep kit</td>
			<td>Promega Corporation</td>
			<td>A1330</td>
			<td></td>
		</tr>
		<tr>
			<td>Maxiprep kit</td>
			<td>Life Technologies Australia</td>
			<td>K2100-07</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Gibco</td>
			<td>11995-073</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Life Technologies Australia</td>
			<td>10099-141</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Life Technologies Australia</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>jetPRIME transfection buffer</td>
			<td>Polyplus</td>
			<td>114-15</td>
			<td></td>
		</tr>
		<tr>
			<td>jetPRIME transfection reagent</td>
			<td>Polyplus</td>
			<td>114-15</td>
			<td></td>
		</tr>
		<tr>
			<td>PhosSTOP (Phosphatase inhibitor)</td>
			<td>Sigma-Aldrich</td>
			<td>4906837001</td>
			<td></td>
		</tr>
		<tr>
			<td>cOmplete, Mini, EDTA-free Protease inhibitor cocktail</td>
			<td>Roche</td>
			<td>11873580001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Scraper</td>
			<td>Sarstedt</td>
			<td>83.1832</td>
			<td></td>
		</tr>
		<tr>
			<td>GFP-Trap_A</td>
			<td>Chromotek Gmbh</td>
			<td>gta-100</td>
			<td>GFP nanobody coupled to agarose beads</td>
		</tr>
		<tr>
			<td>N-terminal GFP monoclonal antibody&#160;</td>
			<td>Covance</td>
			<td>MMS-118P</td>
			<td>Will detect the V1 tag within the BiFC vectors</td>
		</tr>
		<tr>
			<td>C-terminal GFP monoclonal antibody</td>
			<td>Roche</td>
			<td>11814460001</td>
			<td>Will detect the V2 tag within the BiFC vectors</td>
		</tr>
		<tr>
			<td>Sample buffer</td>
			<td>Invitrogen</td>
			<td>NP0008</td>
			<td>Supplemented with 1 mL &#946;-mercaptoethanol.</td>
		</tr>
		<tr>
			<td>Sequencing grade modified trypsin</td>
			<td>Promega Corporation</td>
			<td>V5117h</td>
			<td></td>
		</tr>
		<tr>
			<td>LoBind microcentrifuge tubes</td>
			<td>Point of Care Diagnostics</td>
			<td>0030 108 116</td>
			<td></td>
		</tr>
		<tr>
			<td>Iodoacetamide</td>
			<td>Sigma-Aldrich</td>
			<td>I1149-5G</td>
			<td>Prepared at 5 mg/mL in ultrapure water</td>
		</tr>
		<tr>
			<td>Trifluoroacetic Acid - Sequanal Grade</td>
			<td>Thermo Fisher</td>
			<td>10628494</td>
			<td></td>
		</tr>
		<tr>
			<td>3M Empore solid phase extraction C18 disks (octadecyl) - 4.7 cm</td>
			<td>Thermo Fisher</td>
			<td>14-386-2</td>
			<td>To prepare stage tips, cut 1 mm disks using an appropriately sized hole punch. Alternatively, pre-prepared stage tips can also be purchased, see below.</td>
		</tr>
		<tr>
			<td>C18 Stage Tips, 10 &#181;L bed</td>
			<td>Thermo Fisher</td>
			<td>87782</td>
			<td></td>
		</tr>
		<tr>
			<td>Formic acid OPTIMA for LC/MS grade 50mL</td>
			<td>Thermo Fisher</td>
			<td>FSBA117-50</td>
			<td></td>
		</tr>
		<tr>
			<td>1.9 &#956;m C18 ReproSil particles</td>
			<td>Dr. Maisch GmbH</td>
			<td>r119.aq.</td>
			<td>Stationary phase particles</td>
		</tr>
		<tr>
			<td>Acetonitrile OPTIMA LC/MS grade&#160;</td>
			<td>Thermo Fisher</td>
			<td>FSBA955-4</td>
			<td></td>
		</tr>
		<tr>
			<td>Easy-nLC HPLC&#160;</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LTQ Orbitrap Velos Pro</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8787</td>
			<td>Non-ionic detergent (100%)</td>
		</tr>
		<tr>
			<td>DH5&#945; cells</td>
			<td>Thermo Fisher</td>
			<td></td>
			<td>Heat-shock-competent cells</td>
		</tr>
	</tbody>
</table>

dissecting multi-protein signaling complexes by bimolecular complementation affinity purification (bicap)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research is deciphering how these dynamic protein complexes act to integrate signals from multiple sources in order to direct a specific biological response, and how this becomes deregulated in many disease settings. Despite the importance of this key biochemical mechanism, there is a lack of experimental techniques that can facilitate the specific and sensitive deconvolution of these multi-molecular signaling complexes.
Here this shortcoming is addressed through the combination of a protein complementation assay with a conformation-specific nanobody, which we have termed Bimolecular Complementation Affinity Purification (BiCAP). This novel technique facilitates the specific isolation and downstream proteomic characterization of any pair of interacting proteins, to the exclusion of un-complexed individual proteins and complexes formed with competing binding partners. 
The BiCAP technique is adaptable to a wide array of downstream experimental assays, and the high degree of specificity afforded by this technique allows more nuanced investigations into the mechanics of protein complex assembly than is currently possible using standard affinity purification techniques.

Following the use of recombination cloning to generate of V1 and V2 tagged genes of interest with the BiFC pDEST plasmids, co-transfection of two plasmids containing an interacting pair of proteins will result in the generation of a Venus fluorescent signal after approximately 8-24 hours. In the absence of a positive signal it is possible that the protein interaction may not be occurring due to the choice of cell line, a low transfection efficiency or that the orientation of the BiFC tags...

Watch this Scientific Journal Video about Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP at JoVE.com

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification BiCAP

This manuscript describes the protocol for Bimolecular Complementation Affinity Purification (BiCAP). This novel method facilitates the specific isolation and downstream proteomic characterization of any two interacting proteins, while excluding un-complexed individual proteins as well as complexes formed with competing binding partners.

Dissecting Multi-protein Signaling Complexes by Bimolecular Complementation Affinity Purification (BiCAP)

The assembly of protein complexes is a central mechanism underlying the regulation of many cell signaling pathways. A major focus of biomedical research ...

This method can help us answer key questions in cell biology and biochemistry, such as how the dynamic assembly of protein and protein complexes control signaling pathways and cell behavior. The main advantage of this technique is that we can specifically isolate two interacting proteins, while excluding uncomplexed individual proteins, and competing binding partners. Though we use this method to provide insight into receptive tyrosine kinase dimerization in breast cancer, it can also be applied to any other physiological setting or disease.We first had an idea for this method when we realized the GFP nanobody recognized the three-dimensional epitope of GFP and other fluorescent proteins. To begin, add 150 nanograms of entry vector, 150 nanograms of destination vector, and eight microliters of TE buffer, to a 0.2 milliliter tube. Next add two microliters of recombinase and ZiMex, and centrifuge the two briefly.Incubate the reaction for one hour at room temperature, then add one microliter of Proteinase K solution, and incubate at 37 degrees celsius for 10 minutes to stop the reaction. Next, thaw heat shock-competent cells on ice. And transfer 50 microliters of the cells to a 14 milliliter round-bottomed polypropylene tube.Add one microliter of reaction product to the cells, and mix gently. Incubate the cells for 20 minutes on ice. Then heat shock the cells in a 42 degree celsius water bath for 45 seconds before returning the cells to the ice.Add one milliliter of LB media to the cells. And incubate the sample with shaking for one hour at 37 degrees celsius. Then, plate the transformation onto a 10 centimeter agar plate with ampicillin, and incubate at 37 degrees celsius overnight.To begin plasmid purification, add ampicillin and 100 milliliters of LB media to a large conical flask. Then use an inoculation loop to remove a single colony from the agar plate. Submerge the inoculation loop in LB media and briefly mix, then cover the top of the flask with aluminum foil, and incubate it overnight, at 37 degrees celsius with shaking.From here purify the plasmid DNA using a standard Maxiprep plasmid DNA purification kit, before proceeding with transfection. First, seed HEK 293T cells in 10 centimeter dishes, containing 10 milliliters of DMEM. Then dilute 2.5 micrograms of each bimolecular fluorescence complementation vector in 500 microliters of transfection buffer.Add 10 microliters of the transfection reagent, and vortex the mixture for 10 seconds. Then briefly centrifuge the samples, and incubate them at room temperature for 10 minutes. Next add the DNA transfection mixture dropwise to the dish.Then incubate the samples for eight to 24 hours. Prepare a cell lysis buffer and supplemented cell lysis buffer as outlined in the text protocol. Then wash the cells with ice cold PBS twice.Aspirate the PBS and add one millimeter of ice-cold supplemented cell lysis buffer. Next, place the dish on ice and incubate for five minutes. Then use a cell scraper to remove the cells, and transfer them to a chilled micro-centrifuge tube.Centrifuge the tube at 18, 000 times gravity for five minutes at four degrees celsius, to remove the cellular debris, then transfer the clear supernatant to a fresh micro-centrifuge tube. Wash an appropriate volume of agarose beads in one milliliter of PBS, then centrifuge the beads at 300 times gravity, and carefully remove the supernatant. Next add 20 microliters of agarose beads to each sample.And incubate at four degrees celsius for two hours, with end-to-end rotation. Centrifuge the beads at 300 times gravity and wash them in cell lysis buffer three times. Then re-suspend the washed beads in 50 microliters of diluted sample buffer.Finally, heat the samples at 95 degrees celsius for two to three minutes. In this study, the BiCAP method is used to observe a positive interaction for several protein types approximately 16 hours after co-transfection of HEK293T cells. Con-focal microscopy is used to observe the dimerization of the RTKERBB2 at the plasma membrane, and the binding of Ubiquitin to the E3 ligase UBR5.After affinity purification with the GFB nanobody, only the full length Venus control protein, and interacting V1 and V2 tagged proteins can be detected in the co-transfection sample. While attempting this procedure, remember to optimize the placement of the bi-molecular fluorescence complementation tag, and either the C-terminal or N-terminal end of your protein of interest. This is necessary to observe a positive protein/protein interaction.After watching this video you should have a good understanding of how to perform the BiCAP technique, and how to apply it to your protein interaction of interest. Following this procedure, many of the downstream characterization techniques could be employed, like ChIPseek to answer additional questions, such as how dimerization of the transcription factors controls DNA binding.

dissecting-multi-protein-signaling-complexes-bimolecular

Research

JoVE Journal

Immunology and Infection

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting (FACS)

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known phenotype.  Other bulk methods of purification include panning, complement depletion and magnetic bead separation.  However, FACS has several advantages over other available methods.  FACS is the preferred method when very high purity of the desired population is required, when the target cell population expresses a very low level of the identifying marker or when cell populations require separation based on differential marker density.  In addition, FACS is the only available purification technique to isolate cells based on internal staining or intracellular protein expression, such as a genetically modified fluorescent protein marker.  FACS allows the purification of individual cells based on size, granularity and fluorescence.  In order to purify cells of interest, they are first stained with fluorescently-tagged monoclonal antibodies (mAb), which recognize specific surface markers on the desired cell population (1).  Negative selection of unstained cells is also possible.  FACS purification requires a flow cytometer with sorting capacity and the appropriate software.  For FACS, cells in suspension are passed as a stream in droplets with each containing a single cell in front of a laser.  The fluorescence detection system detects cells of interest based on predetermined fluorescent parameters of the cells.  The instrument applies a charge to the droplet containing a cell of interest and an electrostatic deflection system facilitates collection of the charged droplets into appropriate collection tubes (2).  The success of staining and thereby sorting depends largely on the selection of the identifying markers and the choice of mAb.  Sorting parameters can be adjusted depending on the requirement of purity and yield.  Although FACS requires specialized equipment and personnel training, it is the method of choice for isolation of highly purified cell populations.

Purification of Specific Cell Population by Fluorescence Activated Cell Sorting FACS

For many scientific studies requiring a biological and chemical analysis of cell populations the cells must be in a high state of purity. Fluorescence activated cell sorting (FACS) is a superior method in which to obtain pure cell populations.

Experimental and clinical studies often require highly purified cell populations.  FACS is a technique of choice to purify cell populations of known ...

Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry:  a Novel Use for a Cell Purification Method

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck &amp; Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.

Quantification of cellular inflammation in the injured/pathological CNS by flow cytometry is complicated by lipid/myelin debris that can have similar size and granulation to cells, decreasing sensitivity/accuracy. We have advanced a cell preparation method to remove myelin debris and improve cell detection by flow cytometry in the injured spinal cord.

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true ...

Dissecting Host-virus Interaction in Lytic Replication of a Model Herpesvirus

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral cytokine production1,2. In order to colonize the host, viruses are obligate to evade host antiviral responses and manipulate signaling pathways. Unraveling the host-virus interaction will shed light on the development of novel therapeutic strategies against viral infection.
Murine &gamma;HV68 is closely related to human oncogenic Kaposi's sarcoma-associated herpesvirus and Epsten-Barr virus3,4. &gamma;HV68 infection in laboratory mice provides a tractable small animal model to examine the entire course of host responses and viral infection in vivo, which are not available for human herpesviruses. In this protocol, we present a panel of methods for phenotypic characterization and molecular dissection of host signaling components in &gamma;HV68 lytic replication both in vivo and ex vivo. The availability of genetically modified mouse strains permits the interrogation of the roles of host signaling pathways during &gamma;HV68 acute infection in vivo. Additionally, mouse embryonic fibroblasts (MEFs) isolated from these deficient mouse strains can be used to further dissect roles of these molecules during &gamma;HV68 lytic replication ex vivo.
Using virological and molecular biology assays, we can pinpoint the molecular mechanism of host-virus interactions and identify host and viral genes essential for viral lytic replication. Finally, a bacterial artificial chromosome (BAC) system facilitates the introduction of mutations into the viral factor(s) that specifically interrupt the host-virus interaction. Recombinant &gamma;HV68 carrying these mutations can be used to recapitulate the phenotypes of &gamma;HV68 lytic replication in MEFs deficient in key host signaling components. This protocol offers an excellent strategy to interrogate host-pathogen interaction at multiple levels of intervention in vivo and ex vivo.
Recently, we have discovered that &gamma;HV68 usurps an innate immune signaling pathway to promote viral lytic replication5. Specifically, &gamma;HV68 de novo infection activates the immune kinase IKK&beta; and activated IKK&beta; phosphorylates the master viral transcription factor, replication and transactivator (RTA), to promote viral transcriptional activation. In doing so, &gamma;HV68 efficiently couples its transcriptional activation to host innate immune activation, thereby facilitating viral transcription and lytic replication. This study provides an excellent example that can be applied to other viruses to interrogate host-virus interaction.

We describe a protocol to identify key roles of host signaling molecules in lytic replication of a model herpesvirus, gamma herpesvirus 68 (&gamma;HV68). Utilizing genetically modified mouse strains and embryonic fibroblasts for &gamma;HV68 lytic replication, the protocol permits both phenotypic characterization and molecular interrogation of virus-host interactions in viral lytic replication.

In response to viral infection, a host develops various defensive responses, such as activating innate immune signaling pathways that lead to antiviral ...

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction

Lipopolysaccharide (LPS) is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of lipid A, which is embedded in the outer membrane, a core oligosaccharide and repeating O-antigen units that extend outward from the surface of the cell1, 2. LPS is an immunodominant molecule that is important for the virulence and pathogenesis of many bacterial species, including Pseudomonas aeruginosa, Salmonella species, and Escherichia coli3-5, and differences in LPS O-antigen composition form the basis for serotyping of strains. LPS is involved in attachment to host cells at the initiation of infection and provides protection from complement-mediated killing; strains that lack LPS can be attenuated for virulence6-8. For these reasons, it is important to visualize LPS, particularly from clinical isolates. Visualizing LPS banding patterns and recognition by specific antibodies can be useful tools to identify strain lineages and to characterize various mutants. 
In this report, we describe a hot aqueous-phenol method for the isolation and purification of LPS from Gram-negative bacterial cells. This protocol allows for the extraction of LPS away from nucleic acids and proteins that can interfere with visualization of LPS that occurs with shorter, less intensive extraction methods9. LPS prepared this way can be separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and directly stained using carbohydrate/glycoprotein stains or standard silver staining methods. Many anti-sera to LPS contain antibodies that cross-react with outer membrane proteins or other antigenic targets that can hinder reactivity observed following Western immunoblot of SDS-PAGE-separated crude cell lysates. Protease treatment of crude cell lysates alone is not always an effective way of removing this background using this or other visualization methods. Further, extensive protease treatment in an attempt to remove this background can lead to poor quality LPS that is not well resolved by any of the aforementioned methods. For these reasons, we believe that the following protocol, adapted from Westpahl and Jann10, is ideal for LPS extraction.

We describe a modified hot aqueous-phenol extraction method for purifying lipopolysaccharide (LPS) from Gram-negative bacteria. Once extracted, the LPS can be subsequently analyzed by SDS-PAGE and visualized by direct staining or Western immunoblot.

Lipopolysaccharide (LPS)  is a major component of Gram-negative bacterial outer membranes. It is a tripartite molecule consisting of  lipid A, which is ...

Affinity Purification of Influenza Virus Ribonucleoprotein Complexes from the Chromatin of Infected Cells

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the single-stranded genome is encapsidated by the nucleoprotein (NP), and associated with the trimeric polymerase complex consisting of the PA, PB1, and PB2 subunits. However, in contrast to most RNA viruses, influenza viruses perform viral RNA synthesis in the nuclei of infected cells. Interestingly, viral mRNA synthesis uses cellular pre-mRNAs as primers, and it has been proposed that this process takes place on chromatin1. Interactions between the viral polymerase and the host RNA polymerase II, as well as between NP and host nucleosomes have also been characterized1,2.
Recently, the generation of recombinant influenza viruses encoding a One-Strep-Tag genetically fused to the C-terminus of the PB2 subunit of the viral polymerase (rWSN-PB2-Strep3) has been described. These recombinant viruses allow the purification of PB2-containing complexes, including vRNPs, from infected cells. To obtain purified vRNPs, cell cultures are infected, and vRNPs are affinity purified from lysates derived from these cells. However, the lysis procedures used to date have been based on one-step detergent lysis, which, despite the presence of a general nuclease, often extract chromatin-bound material only inefficiently. 
Our preliminary work suggested that a large portion of nuclear vRNPs were not extracted during traditional cell lysis, and therefore could not be affinity purified. To increase this extraction efficiency, and to separate chromatin-bound from non-chromatin-bound nuclear vRNPs, we adapted a step-wise subcellular extraction protocol to influenza virus-infected cells. Briefly, this procedure first separates the nuclei from the cell and then extracts soluble nuclear proteins (here termed the "nucleoplasmic" fraction). The remaining insoluble nuclear material is then digested with Benzonase, an unspecific DNA/RNA nuclease, followed by two salt extraction steps: first using 150 mM NaCl (termed "ch150"), then 500 mM NaCl ("ch500") (Fig. 1). These salt extraction steps were chosen based on our observation that 500 mM NaCl was sufficient to solubilize over 85% of nuclear vRNPs yet still allow binding of tagged vRNPs to the affinity matrix.
After subcellular fractionation of infected cells, it is possible to affinity purify PB2-tagged vRNPs from each individual fraction and analyze their protein and RNA components using Western Blot and primer extension, respectively. Recently, we utilized this method to discover that vRNP export complexes form during late points after infection on the chromatin fraction extracted with 500 mM NaCl (ch500)3.

Influenza viruses replicate their RNA genome in association with host-cell chromatin. Here, we present a method to purify intact viral ribonucleoprotein complexes from the chromatin of infected cells. Purified viral complexes can be analyzed by both Western blot and primer extension of protein and RNA content, respectively.

Like all negative-strand RNA viruses, the genome of influenza viruses is packaged in the form of viral ribonucleoprotein complexes (vRNP), in which the ...

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe pneumonia "Legionnaires' disease"1. In protozoan and mammalian phagocytes, L. pneumophila employs a conserved mechanism to form a specific, replication-permissive compartment, the "Legionella-containing vacuole" (LCV). LCV formation requires the bacterial Icm/Dot type IV secretion system (T4SS), which translocates as many as 275 "effector" proteins into host cells. The effectors manipulate host proteins as well as lipids and communicate with secretory, endosomal and mitochondrial organelles2-4.
The formation of LCVs represents a complex, robust and redundant process, which is difficult to grasp in a reductionist manner. An integrative approach is required to comprehensively understand LCV formation, including a global analysis of pathogen-host factor interactions and their temporal and spatial dynamics. As a first step towards this goal, intact LCVs are purified and analyzed by proteomics and lipidomics.
The composition and formation of pathogen-containing vacuoles has been investigated by proteomic analysis using liquid chromatography or 2-D gel electrophoresis coupled to mass-spectrometry. Vacuoles isolated from either the social soil amoeba Dictyostelium discoideum or mammalian phagocytes harboured Leishmania5, Listeria6, Mycobacterium7, Rhodococcus8, Salmonella9 or Legionella spp.10. However, the purification protocols employed in these studies are time-consuming and tedious, as they require e.g. electron microscopy to analyse LCV morphology, integrity and purity. Additionally, these protocols do not exploit specific features of the pathogen vacuole for enrichment.
The method presented here overcomes these limitations by employing D. discoideum producing a fluorescent LCV marker and by targeting the bacterial effector protein SidC, which selectively anchors to the LCV membrane by binding to phosphatidylinositol 4-phosphate (PtdIns(4)P)3,11 . LCVs are enriched in a first step by immuno-magnetic separation using an affinity-purified primary antibody against SidC and a secondary antibody coupled to magnetic beads, followed in a second step by a classical Histodenz density gradient centrifugation12,13 (Fig. 1).
A proteome study of isolated LCVs from D. discoideum revealed more than 560 host cell proteins, including proteins associated with phagocytic vesicles, mitochondria, ER and Golgi, as well as several GTPases, which have not been implicated in LCV formation before13. LCVs enriched and purified with the protocol outlined here can be further analyzed by microscopy (immunofluorescence, electron microscopy), biochemical methods (Western blot) and proteomic or lipidomic approaches.

Purification of Pathogen Vacuoles from Legionella-infected Phagocytes

This article describes a method for the isolation and purification of intact Legionella-containing vacuoles (LCVs) from amoeba and macrophages. The two-step protocol comprises LCV enrichment by immuno-magnetic separation using an antibody against a bacterial LCV marker and further purification by density gradient centrifugation.

The opportunistic pathogen Legionella pneumophila is an amoeba-resistant bacterium, which also replicates in alveolar macrophages thus causing the severe ...

Dissecting Innate Immune Signaling in Viral Evasion of Cytokine Production

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. Conversely, viruses have evolved intricate strategies to evade and exploit host immune signaling for survival and propagation. Viral immune evasion, entailing host defense and viral evasion, provides one of the most fascinating and dynamic interfaces to discern the host-virus interaction. These studies advance our understanding in innate immune regulation and pave our way to develop novel antiviral therapies.
Murine &#947;HV68 is a natural pathogen of murine rodents. &#947;HV68 infection of mice provides a tractable small animal model to examine the antiviral response to human KSHV and EBV of which perturbation of in vivo virus-host interactions is not applicable. Here we describe a protocol to determine the antiviral cytokine production. This protocol can be adapted to other viruses and signaling pathways.
Recently, we have discovered that &#947;HV68 hijacks MAVS and IKK&#946;, key innate immune signaling components downstream of the cytosolic RIG-I and MDA5, to abrogate NF&#922;B activation and antiviral cytokine production. Specifically, &#947;HV68 infection activates IKK&#946; and that activated IKK&#946; phosphorylates RelA to accelerate RelA degradation. As such, &#947;HV68 efficiently uncouples NF&#922;B activation from its upstream activated IKK&#946;, negating antiviral cytokine gene expression. This study elucidates an intricate strategy whereby the upstream innate immune activation is intercepted by a viral pathogen to nullify the immediate downstream transcriptional activation and evade antiviral cytokine production.

We describe a protocol to measure the antiviral cytokine production in mice infected with a model herpesvirus, murine gamma herpesvirus 68 (&#947;HV68) that is closely-related to human Kaposi&#8217;s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV). Utilizing genetically modified mouse strains and mouse embryonic fibroblasts (MEFs), we assessed the antiviral cytokine production both in vivo and ex vivo. &#8220;Reconstituting&#8221; the expression of innate immune components in knockout embryonic fibroblasts by lentiviral transduction, we further pinpoint specific innate immune molecules and dissect the key signaling events that differentially regulate the antiviral cytokine production.

In response to a viral infection, the host innate immune response is activated to up-regulate gene expression and production of antiviral cytokines. ...

Rapid and Robust Analysis of Cellular and Molecular Polarization Induced by Chemokine Signaling

Cells respond to chemokine stimulation by losing their round shape in a process called polarization, and by altering the subcellular localization of many proteins. Classic imaging techniques have been used to study these phenomena. However, they required the manual acquisition of many cells followed by time consuming quantification of the morphology and the co-localization of the staining of tens of cells. Here, a rapid and powerful method is described to study these phenomena on samples consisting of several thousands of cells using an imaging flow cytometry technology that combines the advantages of a microscope with those of a cytometer. Using T lymphocytes stimulated with CCL19 and staining for MHC Class I molecules and filamentous actin, a gating strategy is presented to measure simultaneously the degree of shape alterations and the extent of co-localization of markers that are affected by CCL19 signaling. Moreover, this gating strategy allowed us to observe the segregation of filamentous actin (at the front) and phosphorylated Ezrin-Radixin-Moesin (phospho-ERM) proteins (at the rear) in polarized T cells after CXCL12 stimulation. This technique was also useful to observe the blocking effect on polarization of two different elements: inhibition of actin polymerization by a pharmacological inhibitor and expression of mutants of the Par6/atypical PKC signaling pathway. Thus, evidence is shown that this technique is useful to analyze both morphological alterations and protein redistributions.

Chemokine signaling elicits marked alterations of cellular morphology and some important redistributions of intracellular proteins. Here, a rapid and detailed protocol is provided to study these events.

Cells respond to chemokine stimulation by losing their round shape in a process called polarization, and by altering the subcellular localization of many ...

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the viral capsid (CA) core into the cytoplasm. Subsequently, the viral Reverse Transcriptase (RT), as part of a namesake nucleoprotein complex termed the Reverse Transcription Complex (RTC), converts the viral single-stranded RNA genome into a double-stranded DNA copy (vDNA). This leads to the biogenesis of another nucleoprotein complex, termed the pre-integration complex (PIC), composed of the vDNA and associated virus proteins and host factors. The PIC-associated viral integrase (IN) orchestrates the integration of the vDNA into the host chromosomal DNA in a temporally and spatially regulated two-step process. First, the IN processes the 3&#39; ends of the vDNA in the cytoplasm and, second, after the PIC traffics to the nucleus, it mediates integration of the processed vDNA into the chromosomal DNA. The PICs isolated from target cells acutely infected with HIV-1 are functional in vitro, as they are competent to integrate the associated vDNA into an exogenously added heterologous target DNA. Such PIC-based in vitro integration assays have significantly contributed to delineating the mechanistic details of retroviral integration and to discovering IN inhibitors. In this report, we elaborate upon an updated HIV-1 PIC assay that employs a nested real-time quantitative Polymerase Chain Reaction (qPCR)-based strategy for measuring the in vitro integration activity of isolated native PICs.

Measurement of In Vitro Integration Activity of HIV-1 Preintegration Complexes

This report describes an in vitro assay for measuring the human immunodeficiency virus type 1 (HIV-1) preintegration complex (PIC)-associated integration activity in the cytoplasmic extracts of acutely infected cells. The integration of PIC-associated HIV-1 DNA into heterologous target DNA is quantified by using a nested real-time polymerase chain reaction strategy.

HIV-1 envelope proteins engage cognate receptors on the target cell surface, which leads to viral-cell membrane fusion followed by the release of the ...