This work was supported by a grant from the Canadian Institutes for Health Research (MOP-86514).

Experimental procedures with mice were approved by the Animal Care Committee of McGill University (protocol 4966).
1. Preparation of Protein Extracts from Cultured Cells
<ol>
	<li>Wash cultured cells twice with 10&#160;ml of ice-cold phosphate buffered saline (PBS).</li>
	<li>Scrape adherent cells with either a rubber policeman or a plastic cell scraper in 1&#160;ml of ice-cold PBS, transfer suspension into a 1.5&#160;ml microcentrifuge tube.</li>
	<li>Spin in a microcentrifuge for 5&#160;min at 700&#160;x&#160;g, at 4&#160;&#176;C. Aspirate PBS.</li>
	<li>Add 100&#160;&#956;l of ice-cold cytoplasmic lysis buffer (Table 1) per 107 cells, and pipette up and down.</li>
	<li>Incubate on ice for 20&#160;min.</li>
	<li>Spin for 10&#160;min at full speed in a microcentrifuge at 4&#160;&#176;C.</li>
	<li>Discard pellet. Transfer supernatant into new 1.5&#160;ml microcentrifuge tube and keep on ice.</li>
	<li>Determine protein concentration (usually 1 - 10&#160;&#956;g/&#956;l) using the Bradford assay23.</li>
	<li>Aliquot and store cell extracts at -80&#160;&#176;C until use.</li>
</ol>
2. Preparation of Protein Extracts from Mouse Liver and Spleen
<ol>
	<li>Euthanize a mouse with CO2 inhalation.</li>
	<li>Lay the euthanized animal on a clean pad over a dissecting board. Open the abdomen with scissors.</li>
	<li>Dissect the liver and the spleen by using scissors and forceps, and rinse each tissue in approximately 50&#160;ml ice-cold PBS.</li>
	<li>Immediately cut tissues into small pieces with a scalpel (for example: approximately 1 - 2&#160;mm3).</li>
	<li>Without delay, put pieces of tissues in a fresh cryotube and then snap freeze them in liquid nitrogen. Store snap-frozen tissue aliquots at -80&#160;&#176;C until use.</li>
	<li>Homogenize one piece of frozen tissue (approximately 1 - 2&#160;mm3) in 0.25 - 0.5&#160;ml of ice-cold cytoplasmic lysis buffer (Table 1) with a tissue homogenizer for 10&#160;sec.</li>
	<li>Transfer homogenate to 1.5&#160;ml microcentrifuge tube and chill on ice for 20&#160;min.</li>
	<li>Spin for 10&#160;min at full speed in a microcentrifuge at 4&#160;&#176;C.</li>
	<li>Discard pellet and transfer supernatant into new 1.5 ml microcentrifuge tube. Keep on ice.</li>
	<li>Determine protein concentration (usually 1 - 10&#160;&#956;g/&#956;l) using the Bradford assay23.</li>
	<li>Aliquot and store cell extracts at -80&#160;&#176;C until use.</li>
</ol>
3. Preparation of Radiolabeled IRE-probe
<ol>
	<li>Linearize the IRE-containing plasmid I-12.CAT22 by incubating at 37&#160;&#176;C for 1&#160;hr with the restriction endonuclease XbaI (1&#160;U per &#956;g of plasmid), which cleaves downstream of the IRE sequence. The linearized plasmid will be used as template for in vitro transcription.</li>
	<li>Set up an in vitro transcription reaction in a total volume of 20&#160;&#956;l. Use the stock solutions shown in Table 2 and add: 1&#160;&#956;l linearized plasmid template, 4&#160;&#956;l transcription buffer; 1&#160;&#956;l mix of ATP/CTP/GTP mix, 10&#160;&#956;l [&#945;-32P]-UTP, 2&#160;&#956;l dithiothreitol, 1&#160;&#956;l RNase inhibitor and 1&#160;&#956;l T7 RNA polymerase. Mix by pipetting up and down.</li>
	<li>Incubate at 40&#160;&#176;C for 1&#160;hr24.</li>
</ol>
4. Purification of Radiolabeled IRE-probe
<ol>
	<li>Terminate in vitro transcription reaction by adding 1&#160;&#956;l of 0.5&#160;M EDTA, pH&#160;8. Mix by pipetting up and down.</li>
	<li>Add 10&#160;&#956;l of 10&#160;mg/ml tRNA, as carrier for better precipitation. Mix by pipetting up and down.</li>
	<li>Add 82.5&#160;&#956;l of 3&#160;M ammonium acetate. Mix by vortexing.</li>
	<li>Add 273&#160;&#956;l of ethanol. Mix by vortexing.</li>
	<li>Let stand at RT for 5&#160;min.</li>
	<li>Spin for 10&#160;min at full speed in a microcentrifuge at RT. Discard supernatant.</li>
	<li>Wash pellet with 100&#160;&#956;l of 70% ethanol.</li>
	<li>Spin for 10&#160;min at full speed in a microcentrifuge at RT. Discard supernatant.</li>
	<li>Air dry pellet for 10&#160;min.</li>
	<li>Resuspend pellet in 100&#160;&#956;l of double distilled, previously autoclaved H2O.</li>
	<li>Quantify radioactivity in a liquid scintillation counter, aliquot radiolabeled IRE probe and store at -80&#160;&#176;C until use. Frozen aliquots can be used for up to 3&#160;weeks.</li>
</ol>
5. Preparation of a native polyacrylamide gel for EMSA
<ol>
	<li>Assemble the gel (16 x 16&#160;cm) by using 1.5&#160;mm spacers and comb.</li>
	<li>To prepare a 6% native polyacrylamide gel, use the stock solutions shown in Table 3. Mix 7.5&#160;ml of 40% acrylamide:bisacrylamide, 5&#160;ml of 5x TBE and 37.5&#160;ml double distilled H2O.</li>
	<li>Add 0.5&#160;ml of 10% freshly prepared ammonium persulfate (APS) and 25&#160;&#956;l tetramethylethylenediamine (TEMED).</li>
	<li>Immediately pour the acrylamide solution to the gel and let it polymerize. Wait for approximately 30&#160;min.</li>
	<li>Assemble the electrophoresis apparatus with the gel, fill the tanks with 0.5x TBE and connect with the power supply.</li>
</ol>
6. Electrophoretic Mobility Shift Assay
<ol>
	<li>Dilute 25&#160;&#956;g of protein extract from cells or tissues with cytoplasmic lysis buffer (Table 1) in a total volume of in 10&#160;&#956;l (lower protein concentrations may also be used). Keep on ice. If necessary, add 1&#160;&#956;l of 1:4 diluted 2-mercaptoethanol (2-ME) to activate dormant IRP1 (final concentration: 2%)25.</li>
	<li>Dilute the radiolabeled IRE probe in double distilled H2O to 200,000&#160;cpm/&#956;l, heat denature at 95&#160;&#176;C for 1&#160;min, and cool down at RT for at least 5&#160;min.</li>
	<li>Set up an EMSA reaction by adding 1&#160;&#956;l of radiolabeled IRE probe to the protein extract.</li>
	<li>Incubate for 20&#160;min at RT.</li>
	<li>Add 1&#160;&#956;l of 50&#160;mg/ml heparin to the reaction (to inhibit non-specific protein interactions with the probe9) and continue the incubation for another 10&#160;min. Aliquot the stock solution of heparin (50&#160;mg/ml) and store at -80&#160;&#176;C.</li>
	<li>When using long radiolabeled probes (&#62;60 nucleotides), add 1&#160;&#956;l RNase T1 (1&#160;U/&#956;l) and incubate for 10&#160;min at RT to reduce non-specific protein binding to the probe, and to allow better separation of the RNA/protein complex during electrophoresis.</li>
	<li>Add 3&#160;&#956;l of loading buffer (80% glycerol + bromophenol blue), mix and load on the 6% non-denaturing polyacrylamide gel.</li>
	<li>Run the gel for 60&#160;min at 130&#160;V (5&#160;V/cm).</li>
	<li>Transfer the gel onto a large filter paper and dry.</li>
	<li>Expose to a film and develop autoradiography. Exposure time may range from 1&#160;hr (or less) up to O/N.</li>
</ol>

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The authors declare that they have no competing financial interests.

Herein, we describe a protocol that has been developed to study the IRE-binding activities of IRP1 and IRP2, and we show representative data. By using different probes, this protocol can also be adjusted for the study of other RNA-binding proteins. A critical step is the size of the probe. Usage of long probes, which is common when the exact binding site is unknown, can result in RNA/protein complexes that do not migrate differently than the free RNA. In this case, it is advisable to remove unbound RNA by treatment with ...

The EMSA was originally developed to study the association of DNA-binding proteins with target DNA sequences1,2. The principle is similar for RNA/protein interactions3, which is the focus of this article. Briefly, RNA is negatively charged and will migrate towards the anode during non-denaturing electrophoresis in polyacrylamide (or agarose) gels. Migration within the gel depends on the size of the RNA, which is proportional to its charge. Specific binding of a protein to RNA alters its mobility, and the complex migrates slower compared to the free RNA. This is mainly due to an increase in the molecular mass, but also to alterations in the charge and possibly conformation. Utilizing a labeled RNA as probe allows easy monitoring of the &#8220;gel retardation&#8221; or &#8220;bandshift&#8221;. Usage of 32P-labeled RNA probes is very common and offers high sensitivity. The migration of RNA/protein complexes and free RNA are detected by autoradiography. Drawbacks are the short half-life of 32P (14.29 days), the gradual deterioration of the quality of the probe due to radiolysis, the requirement of a radioactivity license and infrastructure for radioactivity work, and potential biosafety concerns. Therefore, alternative non-isotopic methods for labeling the RNA probe have been developed, for instance with fluorophores or biotin, which enable detection by fluorescent or chemiluminescent imaging4,5. Limitations of these methods are the higher cost and often reduced sensitivity compared to isotopic labeling, and the potential of non-isotopic labels to interfere with the RNA/protein interaction. Non-denaturing polyacrylamide gels are suitable for most EMSA applications and are commonly used. On occasion, agarose gels may pose an alternative for the analysis of large complexes.
The major advantage of EMSA is that it combines simplicity, sensitivity, and robustness4. The assay can be completed within a few hours and does not require sophisticated instrumentation. RNA/protein interactions can be detected by EMSA at concentrations as low as 0.1 nM or less, and within a broad range of binding conditions (pH&#160;4.0 - 9.5, monovalent salt concentration 1 - 300&#160;mM, and temperature 0 - 60&#160;&#176;C).
RNA/protein complex formation can also be studied by the filter-binding assay. This is a simple, fast, and inexpensive procedure based on the retention of RNA/protein complexes in a nitrocellulose filter, while a free RNA probe passes through6. Compared to EMSA, it is limited in cases where the RNA probe contains multiple binding sites, or the crude extract contains more than one RNA-binding proteins that bind to the probe at the same site. While multiple RNA/protein interactions will escape detection by the filter-binding assay, they can be readily visualized by EMSA. In some cases, visualization is even possible when two RNA/protein complexes co-migrate (for instance, human IRP1/IRE and IRP2/IRE complexes), by adding an antibody against one of the RNA-binding proteins to the EMSA reaction, yielding further retardation on the gel (&#8220;supershift&#8221;)7.
The EMSA has been widely used to study IRP1 and IRP2, which are post-transcriptional regulators of iron metabolism8-10. They operate by binding to IREs, phylogenetically conserved hairpin structures within the UTRs of several mRNAs11. IREs were first discovered in the mRNAs encoding ferritin12 and transferrin receptor 1 (TfR1)13, proteins of iron storage and uptake, respectively. Later on, IREs were found in erythroid-specific aminolevulinate synthase (ALAS2)14, mitochondrial aconitase15, ferroportin16, divalent metal transporter 1 (DMT1)17, hypoxia inducible factor 2&#945; (HIF2&#945;)18, and other mRNAs19-21. The prototype H- and L-ferritin mRNAs contain one IRE in their 5&#8217; UTR, while TfR1 mRNA contains multiple IREs in its 3&#8217; UTR. IRE/IRP interactions specifically inhibit ferritin mRNA translation by sterically blocking its association of the 43S ribosomal subunit; moreover, they stabilize TfR1 mRNA against endonucleolytic cleavage. IRP1 and IRP2 share extensive sequence similarity and exhibit high IRE-binding activity in iron-starved cells. In iron-replete cells, IRP1 assembles a cubane Fe-S cluster that converts it to cytosolic aconitase at the expense of its IRE-binding activity, while IRP2 undergoes proteasomal degradation. Thus, the IRE/IRP interactions depend upon the cellular iron status, but are also regulated by other signals, such as H2O2, nitric oxide (NO) or hypoxia. Here, we describe the protocol for assessing IRE-binding activity from crude cell and tissue extracts by EMSA. We used a 32P-labeled H-ferritin IRE probe that was generated by in vitro transcription from a plasmid DNA template (I-12.CAT), where the IRE sequence was originally introduced in sense orientation downstream of the T7 RNA polymerase site by cloning of annealed synthetic oligonucleotides 22.

<table border="0" cellpadding="0" cellspacing="0" width="430">
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			<td height="17" style="height:17px;width:195px;">Name of the Materials&#160;</td>
			<td style="width:119px;">Company</td>
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			<td>SIGMA</td>
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			<td>SIGMA</td>
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			<td>BIORAD</td>
			<td>500-0006</td>
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			<td>Thermoscientific</td>
			<td>EPO111</td>
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			<td>Invitrogen</td>
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			<td>Beckman Coulter</td>
			<td align="right">141349</td>
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		<tr height="17">
			<td height="17" style="height:17px;">heparin</td>
			<td>SIGMA</td>
			<td>H0777</td>
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		<tr height="17">
			<td height="17" style="height:17px;">Rnase T1</td>
			<td>Thermoscientific</td>
			<td>EN0541</td>
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			<td height="17" style="height:17px;"></td>
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			<td height="17" style="height:17px;">Name of the Equipments&#160;</td>
			<td>Company</td>
			<td>Catalog Number&#160;</td>
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			<td height="17" style="height:17px;">Tissue Ruptor</td>
			<td>Qiagen</td>
			<td align="right">9001271</td>
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			<td height="17" style="height:17px;">Scintillation counter</td>
			<td>Beckman Coulter</td>
			<td>LS6500</td>
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			<td height="17" style="height:17px;">Protean II xi Cell&#160;&#160;</td>
			<td>BIORAD</td>
			<td>165-1834</td>
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			<td height="17" style="height:17px;">20 wells combs 1.5mm thick</td>
			<td>BIORAD</td>
			<td>165-1868</td>
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		<tr height="17">
			<td height="17" style="height:17px;">1.5 mm spacers</td>
			<td>BIORAD</td>
			<td>165-1849</td>
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		<tr height="17">
			<td height="17" style="height:17px;">PowerPac</td>
			<td>BIORAD</td>
			<td>164-5070</td>
		</tr>
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electrophoretic mobility shift assay (emsa) for the study of rna-protein interactions: the ire/irp example

RNA/protein interactions are critical for post-transcriptional regulatory pathways. Among the best-characterized cytosolic RNA-binding proteins are iron regulatory proteins, IRP1 and IRP2. They bind to iron responsive elements (IREs) within the untranslated regions (UTRs) of several target mRNAs, thereby controlling the mRNAs translation or stability. IRE/IRP interactions have been widely studied by EMSA. Here, we describe the EMSA protocol for analyzing the IRE-binding activity of IRP1 and IRP2, which can be generalized to assess the activity of other RNA-binding proteins as well. A crude protein lysate containing an RNA-binding protein, or a purified preparation of this protein, is incubated with an excess of32 P-labeled RNA probe, allowing for complex formation. Heparin is added to preclude non-specific protein to probe binding. Subsequently, the mixture is analyzed by non-denaturing electrophoresis on a polyacrylamide gel. The free probe migrates fast, while the RNA/protein complex exhibits retarded mobility; hence, the procedure is also called &#8220;gel retardation&#8221; or &#8220;bandshift&#8221; assay. After completion of the electrophoresis, the gel is dried and RNA/protein complexes, as well as free probe, are detected by autoradiography. The overall goal of the protocol is to detect and quantify IRE/IRP and other RNA/protein interactions. Moreover, EMSA can also be used to determine specificity, binding affinity, and stoichiometry of the RNA/protein interaction under investigation.

A radiolabeled IRE probe was prepared, as described in sections 3 and 4 of the protocol. The sequence of the probe was 5&#39;-GGGCGAAUUC GAGCUCGGUA CCCGGGGAUC CUGCUUCAAC AGUGCUUGGA CGGAUCCU-3&#39;; the bolded nucleotides represent an unpaired C residue and the loop, which are critical IRE features. The specific radioactivity of the probe was 4.5 x 109 cpm/&#956;g of RNA.
To assess the effects of iron perturbations on IRE-binding activity, murine RAW...

Watch this Scientific Journal Video about Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example at JoVE.com

Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example

Here we present a protocol to analyze RNA/protein interactions. The electrophoretic mobility shift assay (EMSA) is based on the differential migration of RNA/protein complexes and free RNA during native gel electrophoresis. By using a radiolabeled RNA probe, RNA/protein complexes can be visualized by autoradiography.

Electrophoretic Mobility Shift Assay (EMSA) for the Study of RNA-Protein Interactions: The IRE/IRP Example

RNA/protein interactions are critical for post-transcriptional regulatory pathways. Among the best-characterized cytosolic RNA-binding proteins are iron ...

electrophoretic-mobility-shift-assay-emsa-for-study-rna-protein

Research

JoVE Journal

Biology

53.4K 조회수.  Jewish General Hospital.  여기에서 우리는 RNA / 단백질 상호 작용을 분석하는 프로토콜을 제시한다. 전기 영동 이동성 변화 분석 (EMSA)이 네이티브 겔 전기 영동시 RNA / 단백질 복합체 및 무료 RNA의 차분에 기초하여 이동된다. 방사성 표지 된 RNA 프로브를 사용함으로써, RNA / 단백질 복합체는 오토 라디오 그래피에 의해 시각화 될 수있다. 

비디오: Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Optimized Fibrin Gel Bead Assay for the Study of Angiogenesis

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These steps include: degradation of the basement membrane, proliferation and migration (sprouting) of endothelial cells (EC) into the extracellular matrix, alignment of EC into cords, branching, lumen formation, anastomosis, and formation of a new basement membrane.  Many in vitro assays have been developed to study this process, but most only mimic certain stages of angiogenesis, and morphologically the vessels within the assays often do not resemble vessels in vivo.  Based on earlier work by Nehls and Drenckhahn, we have optimized an in vitro angiogenesis assay that utilizes human umbilical vein EC and fibroblasts.  This model recapitulates all of the key early stages of angiogenesis and, importantly, the vessels display patent intercellular lumens surrounded by polarized EC. EC are coated onto cytodex microcarriers and embedded into a fibrin gel.  Fibroblasts are layered on top of the gel where they provide necessary soluble factors that promote EC sprouting from the surface of the beads.  After several days, numerous vessels are present that can easily be observed under phase-contrast and time-lapse microscopy. This video demonstrates the key steps in setting up these cultures.

This video demonstrates the protocol of an in vitro angiogenesis assay that recapitulates several stages of angiogenesis. Time-lapse images of sprouting, lumen formation, branching and anastomosis - key features of angiogenesis - are shown.

Angiogenesis is a complex multi-step process, where, in response to angiogenic stimuli, new vessels are created from the existing vasculature.  These ...

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

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 ...

The MultiBac Protein Complex Production Platform at the EMBL

Proteomics research revealed the impressive complexity of eukaryotic proteomes in unprecedented detail. It is now a commonly accepted notion that proteins in cells mostly exist not as isolated entities but exert their biological activity in association with many other proteins, in humans ten or more, forming assembly lines in the cell for most if not all vital functions.1,2 Knowledge of the function and architecture of these multiprotein assemblies requires their provision in superior quality and sufficient quantity for detailed analysis. The paucity of many protein complexes in cells, in particular in eukaryotes, prohibits their extraction from native sources, and necessitates recombinant production. The baculovirus expression vector system (BEVS) has proven to be particularly useful for producing eukaryotic proteins, the activity of which often relies on post-translational processing that other commonly used expression systems often cannot support.3 BEVS use a recombinant baculovirus into which the gene of interest was inserted to infect insect cell cultures which in turn produce the protein of choice. MultiBac is a BEVS that has been particularly tailored for the production of eukaryotic protein complexes that contain many subunits.4 A vital prerequisite for efficient production of proteins and their complexes are robust protocols for all steps involved in an expression experiment that ideally can be implemented as standard operating procedures (SOPs) and followed also by non-specialist users with comparative ease. The MultiBac platform at the European Molecular Biology Laboratory (EMBL) uses SOPs for all steps involved in a multiprotein complex expression experiment, starting from insertion of the genes into an engineered baculoviral genome optimized for heterologous protein production properties to small-scale analysis of the protein specimens produced.5-8 The platform is installed in an open-access mode at EMBL Grenoble and has supported many scientists from academia and industry to accelerate protein complex research projects.

Protein complexes catalyze key cellular functions. Detailed functional and structural characterization of many essential complexes requires recombinant production. MultiBac is a baculovirus/insect cell system particularly tailored for expressing eukaryotic proteins and their complexes. MultiBac was implemented as an open-access platform, and standard operating procedures developed to maximize its utility.

Proteomics  research revealed the impressive complexity of eukaryotic proteomes in  unprecedented detail. It is now a commonly accepted notion that ...

Budding Yeast Protein Extraction and Purification for the Study of Function, Interactions, and Post-translational Modifications

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously hard to disrupt. Here we describe the use of a bead mill homogenizer for the extraction of proteins into buffers optimized to maintain the functions, interactions and post-translational modifications of proteins. Logarithmically growing cells expressing the protein of interest are grown in a liquid growth media of choice. The growth media may be supplemented with reagents to induce protein expression from inducible promoters (e.g. galactose), synchronize cell cycle stage (e.g. nocodazole), or inhibit proteasome function (e.g. MG132). Cells are then pelleted and resuspended in a suitable buffer containing protease and/or phosphatase inhibitors and are either processed immediately or frozen in liquid nitrogen for later use. Homogenization is accomplished by six cycles of 20 sec&#160;bead-beating (5.5 m/sec), each followed by one minute incubation on ice. The resulting homogenate is cleared by centrifugation and small particulates can be removed by filtration. The resulting cleared whole cell extract (WCE) is precipitated using 20% TCA for direct analysis of total proteins by SDS-PAGE followed by Western blotting. Extracts are also suitable for affinity purification of specific proteins, the detection of post-translational modifications, or the analysis of co-purifying proteins. As is the case for most protein purification protocols, some enzymes and proteins may require unique conditions or buffer compositions for their purification and others may be unstable or insoluble under the conditions stated. In the latter case, the protocol presented may provide a useful starting point to empirically determine the best bead-beating strategy for protein extraction and purification. We show the extraction and purification of an epitope-tagged SUMO E3 ligase, Siz1, a cell cycle regulated protein that becomes both sumoylated and phosphorylated, as well as a SUMO-targeted ubiquitin ligase subunit, Slx5.

The preparation of high quality yeast cell extracts is a necessary first step in the analysis of individual proteins or entire proteomes. Here we describe a fast, efficient, and reliable homogenization protocol for budding yeast cells that has been optimized to preserve protein functions, interactions, and post-translational modifications.

Homogenization by bead beating is a fast and efficient way to release DNA, RNA, proteins, and metabolites from budding yeast cells, which are notoriously ...

Combining Single-molecule Manipulation and Imaging for the Study of Protein-DNA Interactions

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method offers the opportunity of investigating interactions occurring in solution (thus avoiding problems due to closeby surfaces as in other single molecule methods), controlling the DNA extension and tracking interaction dynamics as a function of both mechanical parameters and DNA sequence. The methods for establishing successful optical trapping and nanometer localization of single molecules are illustrated. We illustrate the experimental conditions allowing the study of interaction of lactose repressor (lacI), labeled with Atto532, with a DNA molecule containing specific target sequences (operators) for LacI binding. The method allows the observation of specific interactions at the operators, as well as one-dimensional diffusion of the protein during the process of target search. The method is broadly applicable to the study of protein-DNA interactions but also to molecular motors, where control of the tension applied to the partner track polymer (for example actin or microtubules) is desirable.

Here we describe the instrumentation and methods for detecting single fluorescently-labeled protein molecules interacting with a single DNA molecule suspended between two optically trapped microspheres.

The paper describes the combination of optical tweezers and single molecule fluorescence detection for the study of protein-DNA interaction. The method ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay (EMSA) and DNA-affinity Precipitation Assay (DAPA)

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical disease or phenotype. For many diseases and traits, most variants are non-coding, and are thus likely to act by impacting subtle, comparatively hard to predict mechanisms controlling gene expression. Here, we describe a general strategic approach to prioritize non-coding variants, and screen them for their function. This approach involves computational prioritization using functional genomic databases followed by experimental analysis of differential binding of transcription factors (TFs) to risk and non-risk alleles. For both electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genetic variants, a synthetic DNA oligonucleotide (oligo) is used to identify factors in the nuclear lysate of disease or phenotype-relevant cells. For EMSA, the oligonucleotides with or without bound nuclear factors (often TFs) are analyzed by non-denaturing electrophoresis on a tris-borate-EDTA (TBE) polyacrylamide gel. For DAPA, the oligonucleotides are bound to a magnetic column and the nuclear factors that specifically bind the DNA sequence are eluted and analyzed through mass spectrometry or with a reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blot analysis. This general approach can be widely used to study the function of non-coding genetic variants associated with any disease, trait, or phenotype.

Screening for Functional Non-coding Genetic Variants Using Electrophoretic Mobility Shift Assay EMSA and DNA-affinity Precipitation Assay DAPA

We present a strategic plan and protocol for identifying non-coding genetic variants affecting transcription factor (TF) DNA binding. A detailed experimental protocol is provided for electrophoretic mobility shift assay (EMSA) and DNA affinity precipitation assay (DAPA) analysis of genotype-dependent TF DNA binding.

Population and family-based genetic studies typically result in the identification of genetic variants that are statistically associated with a clinical ...