We thank EUROSARF for sending us yeast TAP expression plasmids. We are also grateful for editorial help from Ryan Labadens. This work was supported by a NIH/NINDS grant (R01NS070962) to C.W.

1. Generate UAS-TAP-tagged Transgenic Flies
<ol>
	<li>Generate pUAST-TAP-tagged DNA constructs.
	<ol>
		<li>Decide which side (N- or C-terminus) of the bait protein the TAP tag should be fused to, based on the protein&#39;s structure/function. See discussion for more details.</li>
		<li>Subclone the cDNA coding region of the gene of interest into the multiple cloning sites (MCS) of the pUAST-NTAP or pUAST-CTAP vectors to generate N- or C-terminal-tagged UAS-TAP transgenes, respectively. See Figure 1 for detailed maps and usable restriction sites and reading frames.</li>
	</ol>
	</li>
	<li>Generate UAS-TAP-tagged transgenic flies.
	<ol>
		<li>Generate transgenic flies following standard protocols using P-element-mediated insertion11. A number of injection services are commercially available.</li>
		<li>Cross neuronal GAL4 driver (i.e. BG380-GAL4) to each individual transgenic line and determine the protein expression levels of each line by western blot (with Peroxidase Anti-Peroxidase antibody) and/or immunostaining (with anti-TAP antibody). In general, a transgenic line with a protein expression level that is close to the endogenous protein is recommended for &#160;TAP procedures. See discussion for more details.</li>
		<li>PerformGAL4/UAS-based rescue experiments to confirm the functionality of the TAP-tagged transgenes if loss-of-function mutants of the genes of interest are available. Choose a transgene that can substantially rescue the mutant phenotypes for the following TAP experiments.</li>
	</ol>
	</li>
</ol>
2. Prepare Samples for TAP Procedure
<ol>
	<li>Generate a fly stock that carries both a neuronal GAL4 driver (e.g. BG380-Gal4) and the chosen TAP-tagged transgene in order to ease expansion of fly samples. Collect the F1 progenies of the GAL4 driver and the UAS-transgene cross in rare cases when the above combination causes survival and growth disadvantage.</li>
	<li>Collect small scale samples and optimize lysis condition for solubilizing the TAP-tagged protein.
	<ol>
		<li>Make a series of lysis buffers using a combination of the nonionic detergents NP-40 (0.1-1%), NaDOC (0.1-1%) and Triton X-100 (0.05-0.5%). See Table 1 and discussion for more information.</li>
		<li>On top of a CO2 pad, use #5 forceps to dissect 20 adult heads from the TAP transgene expressing flies and collect them in a 1.5 ml tube to test each lysis buffer condition.</li>
		<li>Add 100 ul testing lysis buffer to the tube and homogenize the heads by stroking up and down with a plastic pestle, then add another 100 ul testing buffer.</li>
		<li>Spin the head lysate at 21,500 x g for 10 min (4 &#176;C), and separate the supernatant and pellet after centrifugation. Add 25 ul 2x&#160;SDS loading buffer to the pellet and 10 ul 4x&#160;SDS loading buffer to 30 ul of supernatant respectively.</li>
		<li>Boil the two samples for 5 min and analyze them side-by-side using SDS-PAGE and subsequent Western Blot with the PAP antibody. Determine the solubility by the ratio of TAP-protein levels in supernatant vs&#160;the pellet.</li>
	</ol>
	</li>
	<li>Prepare sample in large scale
	<ol>
		<li>Expand and collect adult flies.
		<ol>
			<li>Expand the neuronal-GAL4/UAS-TAP-transgene stock in bottles and flip the bottles every 3 days till accumulative 250 bottles are used for collection.</li>
			<li>Collect 1-3 days old adult flies into 50 ml conical tubes, put the tube in liquid nitrogen immediately to deep freeze the flies. &#160;Store the flies in a -80 &#176;C freezer. Note that the volume of the flies must not exceed 2/3 of the 50 ml tube.</li>
		</ol>
		</li>
		<li>Collect fly heads (perform this step on top of powdered dry ice).
		<ol>
			<li>Take out the prechilled sieves and the mortar and pestle from the -80 &#176;C freezer and put them on dry ice, ideally inside a large ice bucket. Stack two U.S.A. standard test sieves with a No. 25 on the top and No. 40 at the bottom.</li>
			<li>Take the frozen flies out and drop them in liquid nitrogen and keep the flies in there for about 10 min.&#160; Vortex or shake the tubes vigorously to break the heads, legs, and wings from the bodies.</li>
			<li>Pour the mixture to the top sieve, and then shake the sieves vigorously while holding both sieves together. After sieving, the bodies will stay on the top sieve, fly heads will be retained on the bottom sieve, and legs, wings, and other debris will fall to the dry ice. Separate the two sieves and carefully transfer the fly heads to the cold mortar.</li>
		</ol>
		</li>
		<li>Homogenize the fly heads
		<ol>
			<li>On top of dry ice, grind the heads with the mortar and the pestle to powdery particles, then transfer the powder to a 15 ml glass Dounce Tissue Grinder that was prechilled&#160;on ice.</li>
			<li>Measure the weights of the grinder before and after the head sample was poured into it, and then calculate how much the head sample weighs. A total of 6-15 grams of fly heads will be sufficient for each TAP experiment adjusting accordingly to the expression levels of the protein. Add 15 ml of ice-cold homogenization buffer (lysis buffer optimized in step 2.2) to the powder and then stroke with the large clearance pestle until it is easy for the pestle to go up and down. Keep the glass grinder on ice at all times.</li>
		</ol>
		</li>
		<li>Prepare the supernatant for TAP
		<ol>
			<li>Transfer the homogenate to a high-speed centrifuge tube and spin for 20 min at ~50,000 x g (4 &#176;C). Transfer the supernatant to a new high-speed centrifuge tube and repeat the centrifugation one more time.</li>
			<li>Transfer the supernatant to an ultracentrifuge tube and perform a 40&#160;min ~250,000 x g spin to further clear the supernatant. The supernatant is ready for the tandem affinity purification procedures after ultracentrifugation.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
3. TAP Purification
The following sections were derived from the S&#233;raphin lab TAP protocol12 (http://web.as.uky.edu/Biology/faculty/rymond/BIO%20510/Bertran%20Seraphin%27s%20TAP%20page.pdf )
<ol>
	<li>Perform IgG bead affinity purification
	<ol>
		<li>Prepare IgG sepharose bead while the samples are being centrifuged. Wash 400 &#181;l IgG bead 3x in a 15 ml Falcon tube with 10 ml cold IgG washing buffer. For each wash, rock the tube gently for 2 min, and then spin down the beads at 1,000 x g for another 2 min. At the end of the third wash, remove the buffer and leave only the beads in the tube.</li>
		<li>Carefully transfer the cleared supernatant (~15 ml) into the 15 ml tube containing the IgG beads. Incubate the beads and brain lysate mix at 4 &#186;C on a nutator for 2 hr.</li>
		<li>Set up a clean and empty micro column with about 15 ml total volume in the cold room. Load the IgG bead mixture by steadily pouring the mix into the column; try not to trap any air bubbles inside the column. Allow the beads to settle in the column and the buffer to slowly drain by gravity flow.</li>
		<li>Wash the column thoroughly with 10 ml of cold IgG washing buffer after all of the brain lysate has flowed through the settled IgG column. Repeat the wash 2x. Note: never let the bead dry in the air.</li>
	</ol>
	</li>
	<li>Perform TEV cleavage
	<ol>
		<li>After the third wash, wash the column again with 10 ml TEV cleavage buffer. This step prepares the IgG bead that sequestrates the bait complex for TEV cleavage.</li>
		<li>Right before the last drop of TEV buffer is about to drip out, put a cap at the bottom of the column to block the flow, add 1.3 ml TEV buffer containing 130 units of TEV enzyme to the column, and then securely cap the top of the column. Make sure the column is sealed well at both ends.</li>
		<li>Rotate the column at 18&#160;&#186;C for 2 hr to allow the TEV enzyme to cleave the peptide at the TEV site and release the protein complex while leaving behind the protein A domain peptide bound to the IgG sepharose beads.</li>
	</ol>
	</li>
	<li>Perform Calmodulin bead affinity purification
	<ol>
		<li>Prepare the Calmodulin beads while the IgG beads are incubated with the TEV enzyme. Wash 200 &#956;l Calmodulin beads in a 15 ml Falcon tube 3x, each time with 10 ml of cold Calmodulin binding buffer. For each wash, gently rock the tube for 2 min on a nutator, and then spin down the beads at 1,000 x g for 2 min. At the end of the third wash, take out all the buffer and leave only the beads in the tube.</li>
		<li>At the end of the TEV incubation (step 3.2.3), return the IgG column back to the cold room and set it straight up. Let the beads settle for 10 min.</li>
		<li>Remove the top cap and then the bottom cap, and then collect the 1.3 ml TEV cleavage product in a 15 ml Falcon tube. Let the buffer drain completely. Add an additional 200 &#181;l TEV buffer to the column to push out the dead volume of the column, collect the flow-out in the same tube.</li>
		<li>Add 4.5 ml of Calmodulin binding buffer and 4.5 &#181;l 1 M CaCl2 to the 1.5 ml TEV cleavage product collected above. The CaCl2 serves to titrate the EDTA in the TEV buffer. Transfer the 6 ml mixture to the tube containing the Calmodulin beads and rotate the tube at 4 &#186;C on a nutator for 1 hr.</li>
		<li>Set up another clean and empty micro column with about 10 ml total volume in the cold room. Load the Calmodulin bead mixture to the column and allow it to drain by gravity.</li>
		<li>When all the solution has flowed through the settled Calmodulin column, wash the column two times, each with 10 ml of cold Calmodulin binding buffer. Note: avoid disturbing the Calmodulin beads and try to keep the surface of the beads as flat as possible during the wash.</li>
	</ol>
	</li>
	<li>Elute the bait complex from Calmodulin column. 
	Right after washing, elute the Calmodulin column with five fractions of 200 &#181;l cold Calmodulin elution buffer. For each fraction, gently add 200 &#181;l of elution buffer to the column and collect the eluate with a marked 1.5 ml Eppendorf tube. Repeat this 4x.</li>
	<li>Analyze the protein complex by SDS-PAGE
	<ol>
		<li>Take a small aliquot from each of the five fractions (about 30 &#181;l) and add SDS loading buffer. Boil the samples for 5 min and load the samples side-by-side with protein molecular markers in a gradient (4-15%) SDS-PAGE gel.</li>
		<li>After the samples have fully resolved in the gel, stop the electrophoresis and stain the gel with any G-250-based sensitive colloidal Coomassie staining procedures such as &#39;blue silver&#39; staining13. Silver staining is optional but not preferable because it is not fully compatible with the subsequent mass spectrometry analysis.&#160; Store the rest of the eluate in a -80 &#176;C freezer for further analysis such as mass spectrometry for uncovering the molecular identities of the purified protein complex. See discussion.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Tandem affinity purification (TAP) method offers a dual purification protocol that allows the isolation and enrichment of protein complexes through two independent affinity purification steps. The design of the TAP tag is not restricted to what is presented in this protocol, other protein binding domains and motifs are also applicable if buffer conditions are adjusted accordingly. A good example of other TAP tags is the GS-TAP tag, a combination of a G protein and a streptavidin-binding motif, designed by Giulio Superti-...

Defining the molecular pathways or networks that mediate a particular biological process is one of the ultimate goals of biomedical research. Fly geneticists have depended heavily on forward genetics, especially modifier genetic screens (both enhancer and suppressor screens), to identify factors that work together, in parallel with, or upstream or downstream of a gene of interest. However, forward genetics screens often times fail to identify essential genes which, when mutated, cause lethality at early developmental stages, or genes with functional redundancy and compensation whose loss of function only cause subtle defects that are hard to score. One way to overcome this difficulty is to screen for direct protein-protein interactions. For more than a decade, a growing list of biochemical methods, including yeast two-hybrid, phage display, chemical cross-linking, Co-IP, Tandem Affinity Purification (TAP), etc.&#160;have been used to investigate protein-protein interactions. Each of these approaches has its own set of strengths and weaknesses in regards to sensitivity and specificity.&#160; Among them, the TAP method allows for detection of physical interaction under near-physiological conditions, &#160;preserves specificity and consistency2&#160;and includes the ability to extend to high-throughput analyses3,4.
The TAP method was originally developed in yeast by Rigautand colleagues1. In this method, a protein of interest is expressed with a TAP tag. The TAP tag harbors two independent affinity-binding domains: a Protein A domain that binds to IgG and a calmodulin-binding domain. The two domains are separated by a TEV (Tobacco Etch Virus) cleavage site. Such a combination allows for two independent rounds of affinity purifications to sufficiently reduce nonspecific bindings and enrich specific bindings1. For this instance, the TAP method is a very powerful method to identify in vivo interactions of a given protein, although overexpressing the exogenous protein may make it more prone to associate with proteins that normally don&#39;t complex with its endogenous counterpart. Since its development, the TAP method has been applied in many other systems, including cell-culture-based systems5,6 and other in vivo model systems6-9. Here we describe the adaptation of the TAP method in Drosophila. We first generate pUAST-NTAP and pUAST-CTAP vectors to facilitate cloning and fusion of the TAP tag to either the N- or C-terminal of the gene of interest. The UAS-TAP-tagged transgene is then expressed in the nervous system under the control of a neuronal GAL4 driver10. Next, a large number of adult fly heads will be collected, which have high content of neural cells and are easy to separate from other body parts after freezing based on size differences. The adult heads are homogenized and cleared by sequential centrifugations, and the supernatant is subject to a TAP procedure described below.

<table border="0" cellpadding="0" cellspacing="0" width="697">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:307px;">U.S.A. standard test sieve No. 25</td>
			<td style="width:104px;">Fisher Scientific&#160;</td>
			<td style="width:119px;">04-881-18&#160;</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">U.S.A. standard test sieve No. 40</td>
			<td style="width:104px;">Fisher Scientific&#160;</td>
			<td style="width:119px;">04-881-21</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#160;Kontes Dounce Tissue Grinders 15 ml</td>
			<td>Kimble Chase</td>
			<td>885300-0015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;"></td>
			<td style="width:104px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">IgG sepharose beads</td>
			<td>Pharmacia</td>
			<td style="width:119px;">17-0969-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Econo-column 0.7 cm x 20 cm</td>
			<td style="width:104px;">Bio-Rad</td>
			<td style="width:119px;">737-4721</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Econo-column 0.5 cm x 15 cm</td>
			<td style="width:104px;">Bio-Rad</td>
			<td style="width:119px;">737-4716</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calmodulin beads</td>
			<td style="width:104px;">Stratagene</td>
			<td style="width:119px;">214303</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Coors Mortar and Pestle</td>
			<td style="width:104px;">CoorsTek</td>
			<td>60311</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">AcTEV Protease</td>
			<td style="width:104px;">Invitrogen</td>
			<td style="width:119px;">12575-015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease Inhibitor Cocktail</td>
			<td style="width:104px;">Roche</td>
			<td style="width:119px;">11836153001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protease Inhibitor Mix</td>
			<td style="width:104px;">Sigma</td>
			<td style="width:119px;">P8340</td>
			<td></td>
		</tr>
	</tbody>
</table>

identifying protein-protein interaction in drosophila adult heads by tandem affinity purification (tap)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. However, the use of biochemical screens aimed at extending the knowledge gained from genetic analysis was explored only recently. Here we describe a method to purify the protein complex that associates with any protein of interest from adult fly heads. This method takes advantage of the Drosophila GAL4/UAS system to express a bait protein fused with a Tandem Affinity Purification (TAP) tag in fly neurons in vivo, and then implements two rounds of purification using a TAP procedure similar to the one originally established in yeast1 to purify the interacting protein complex. At the end of this procedure, a mixture of multiple protein complexes is obtained whose molecular identities can be determined by mass spectrometry. Validation of the candidate proteins will benefit from the resource and ease of performing loss-of-function studies in flies. Similar approaches can be applied to other fly tissues. We believe that the combination of genetic manipulations and this proteomic approach in the fly model system holds tremendous potential for tackling fundamental problems in the field of neurobiology and beyond.

Here we demonstrate our effort in identifying Highwire-interacting proteins in the fly brain. Highwire (Hiw) and its vertebrate and invertebrate homologues are huge ubiquitin ligases that regulate the development and repair of the nervous system14. They share a number of highly conserved functional domains.&#160;However, their molecular actions are not entirely clear. Work done in worm, fly and mouse led to the current working model that Hiw functions as an E3 ligase and as a scaffolding protein to facilitate ...

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Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Drosophila is famous for its powerful genetic manipulation, but not for its suitability of in-depth biochemical analysis. Here we present a TAP-based procedure to identify interacting partners of any protein of interest from the fly brain. This procedure can potentially lead to new avenues of research.

Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification (TAP)

Genetic screens conducted using Drosophila melanogaster (fruit fly) have made numerous milestone discoveries in the advance of biological sciences. ...

identifying-protein-protein-interaction-drosophila-adult-heads-tandem

Research

JoVE Journal

Biology

13.8K Aufrufe.  Louisiana State University Health Sciences Center.  Drosophila ist berühmt für seine leistungsstarken genetischen Manipulation, aber nicht für die Eignung der eingehenden biochemischen Analyse. Hier präsentieren wir eine TAP-basierte Verfahren, um Interaktionspartner eines Proteins von Interesse aus dem Fliegenhirn zu identifizieren. Dieser Vorgang kann möglicherweise zu neue Wege der Forschung führen. 

Serie: Identifying Protein-protein Interaction in Drosophila Adult Heads by Tandem Affinity Purification TAP

Orthogonal Protein Purification Facilitated by a Small Bispecific Affinity Tag

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a demand for general methods that do not require target protein specific optimization1 . To achieve this, purification tags that genetically can be fused to the gene of interest are commonly used2 . The most widely used affinity handle is the hexa-histidine tag, which is suitable for purification under both native and denaturing conditions3 . The metabolic burden for producing the tag is low, but it does not provide as high specificity as competing affinity chromatography based strategies1,2.
Here, a bispecific purification tag with two different binding sites on a 46 amino acid, small protein domain has been developed. The albumin-binding domain is derived from Streptococcal protein G and has a strong inherent affinity to human serum albumin (HSA). Eleven surface-exposed amino acids, not involved in albumin-binding4 , were genetically randomized to produce a combinatorial library. The protein library with the novel randomly arranged binding surface (Figure 1) was expressed on phage particles to facilitate selection of binders by phage display technology. Through several rounds of biopanning against a dimeric Z-domain derived from Staphylococcal protein A5, a small, bispecific molecule with affinity for both HSA and the novel target was identified6 .
The novel protein domain, referred to as ABDz1, was evaluated as a purification tag for a selection of target proteins with different molecular weight, solubility and isoelectric point. Three target proteins were expressed in Escherishia coli with the novel tag fused to their N-termini and thereafter affinity purified. Initial purification on either a column with immobilized HSA or Z-domain resulted in relatively pure products. Two-step affinity purification with the bispecific tag resulted in substantial improvement of protein purity. Chromatographic media with the Z-domain immobilized, for example MabSelect SuRe, are readily available for purification of antibodies and HSA can easily be chemically coupled to media to provide the second matrix.
This method is especially advantageous when there is a high demand on purity of the recovered target protein. The bifunctionality of the tag allows two different chromatographic steps to be used while the metabolic burden on the expression host is limited due to the small size of the tag. It provides a competitive alternative to so called combinatorial tagging where multiple tags are used in combination1,7.

A novel and highly efficient two-step affinity chromatography protocol has been developed and is described in detail. The method is based on a small purification tag with two inherent affinities and is applicable to a wide range of target proteins with different properties.

Due to the high costs associated with purification of recombinant proteins the protocols need to be rationalized. For high-throughput efforts there is a ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

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 ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Purification of Transcripts and Metabolites from Drosophila Heads

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.

Purification of Transcripts and Metabolites from Drosophila Heads

We describe here the procedures for the extraction and purification of mRNA and metabolites from Drosophila heads. We are applying these techniques to better understand the cellular perturbations underlying neuronal degeneration. These methodologies can be easily scaled and adapted for other "omic" projects.

For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Identifying Protein-protein Interaction Sites Using Peptide Arrays

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may result in disease, making protein interactions important drug targets. It is thus highly important to understand these interactions at the molecular level. Protein interactions are studied using a variety of techniques ranging from cellular and biochemical assays to quantitative biophysical assays, and these may be performed either with full-length proteins, with protein domains or with peptides. Peptides serve as excellent tools to study protein interactions since peptides can be easily synthesized and allow the focusing on specific interaction sites. Peptide arrays enable the identification of the interaction sites between two proteins as well as screening for peptides that bind the target protein for therapeutic purposes. They also allow high throughput SAR studies. For identification of binding sites, a typical peptide array usually contains partly overlapping 10-20 residues peptides derived from the full sequences of one or more partner proteins of the desired target protein. Screening the array for binding the target protein reveals the binding peptides, corresponding to the binding sites in the partner proteins, in an easy and fast method using only small amount of protein.
In this article we describe a protocol for screening peptide arrays for mapping the interaction sites between a target protein and its partners. The peptide array is designed based on the sequences of the partner proteins taking into account their secondary structures. The arrays used in this protocol were Celluspots arrays prepared by INTAVIS Bioanalytical Instruments. The array is blocked to prevent unspecific binding and then incubated with the studied protein. Detection using an antibody reveals the binding peptides corresponding to the specific interaction sites between the proteins.

Peptide array screening is a high throughput assay for identifying protein-protein interaction sites. This allows mapping multiple interactions of a target protein and can serve as a method for identifying sites for inhibitors that target a protein. Here we describe a protocol for screening and analyzing peptide arrays.

Protein-protein interactions mediate most of the processes in the living cell and control homeostasis of the organism. Impaired protein interactions may ...

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 ...