We would like to thank Dr. S. Ressler (Department of Molecular and Cellular Biology, Baylor College of Medicine) for suggesting the use of protein concentrators to increase the concentration of MMPs in cell culture supernatants.
This project was supported by grants from the Mrs. Clifford Elder White Graham Endowed Research Fund and the NIH/NIAMS (AR059838) to CB. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

1. Loading and Running the Gel
<ol>
	<li>All samples must be prepared adequately to maintain the function of the enzymes and used immediately after collection or stored frozen at -80&deg;C. The samples must not contain reducing agents (such a &beta;-mercaptoethanol) or be boiled before gel loading.</li>
	<li>Open the pouch containing a gel inside a cassette, rinse the cassette with deionized water.
	<ul>
		<li>Remove the protective tape from the bottom of the cassette and the comb from the top of the gel.</li>
		<li>Rinse the wells three times with running buffer (diluted to 1X in deionized water).</li>
		<li>Place the gel into the Mini-Cell, ensuring that the smaller side of the cassette faces inwards. Lock into place with the Gel Tension Wedge. The Mini-Cell allows to run one or two gels in parallel.</li>
		<li>Fill the top (inside) chamber with 1X running buffer above wells level, check for any leaks. In case of leaks, remove the buffer and reposition the gel.</li>
		<li>Fill the lower chamber with 1X running buffer.</li>
	</ul>
 </li>
	<li>Load 10 &mu;L of protein molecular marker in one well. 
	<ul>
		<li>Mix an equal amount of gel-loading buffer and of sample and load into the wells of the gel using gel-loading tips (changed between each sample). These wells can be loaded with up to 20 &mu;L total.</li>
	</ul>
 </li>
	<li>Place the lid on the Mini-Cell and connect the electrode cords to the power supply. Switch the power supply on and set it to run at 125 V constant for 90 minutes. Check the formation of small bubbles on the wire of the lower chamber, indicating current circulation.</li>
	<li>When running your first gel, monitor progress of the migration every 15 minutes, using the bromophenol blue included in the loading buffer as an indicator. Let the gel run until the indicator dye reaches the bottom of the gel.</li>
</ol>
2. Renaturing and Developing the Gel
<ol>
	<li>For each gel, prepare 100 mL of 1X renaturing buffer and 200 mL of denaturing buffer, both in deionized water.</li>
	<li>When the bromophenol blue tracking dye reaches the bottom of the gel, switch the power supply off, open the Mini-Cell, and remove the gel. Separate the two sides of the cassette using the gel knife (or a weighing spatula). Cut a corner to mark the gel's direction.</li>
	<li>Carefully remove the gel from the cassette and place into a container with 100 mL renaturing buffer. Incubate for 30 minutes at room temperature with gentle agitation.</li>
	<li>Remove the renaturing buffer and add 100 mL of developing buffer to the gel. Incubate for 30 minutes at room temperature with gentle agitation.</li>
	<li>Remove the developing buffer and add 100 mL more of developing buffer to the gel. Incubate overnight (16-18 hours) at 37&deg;C.</li>
	<li>Remove the developing buffer and rinse three times (5 minutes each) with deionized water under gentle agitation at room temperature.</li>
	<li>Scan the gel to save the exact positioning of the protein standard bands as they will become less or not visible after gel staining.</li>
	<li>Stain the gel by adding 20 mL of SimplyBlue Safestain to the gel. Incubate for 1 hour at room temperature under gentle agitation.</li>
	<li>Remove the SimplyBlue SafeStain and de-stain the gel in 100 mL or more deionized water for one hour at room temperature under gentle agitation.</li>
	<li>For better results, replace with fresh deionized water and incubate for another hour or more at room temperature under gentle agitation.</li>
</ol>
3. Data Analysis
<ol>
	<li>Carefully remove the gel from the water and place in a plastic sheet protector.</li>
	<li>Scan the gel with a resolution of 300 dpi or higher. Save the image in the TIFF format (Figure 1A).</li>
	<li>Measure band intensities with ImageJ (or another similar software). 
	<ul>
		<li>Open the TIFF file in ImageJ (Figure 1A).</li>
		<li>Visualize in black and white by selecting "Image&gt;Type&gt;8-bit" (Figure 1B).</li>
		<li>Use the rectangular selection tool to outline the first band, drawing a rectangle at least twice higher than it is wide (this is a software requirement).</li>
		<li>Press "1" or select "Analyze&gt;Gels&gt;Select first lane" and the band will be outlined.</li>
		<li>A new rectangle will appear, move it to the next band and select "Analyze>Gels>Select next lane". Repeat until all bands are selected (Figure 1B).</li>
		<li>Press "3" or select "Analyze>Gels>Plot lanes" to generate the profile plot for each band (Figure 1C).</li>
		<li>Use the straight line selection (should already be automatically selected) to draw base lines so that the peak of interest is a completely enclosed area.</li>
		<li>Select the wand tool and click inside each peak to select it.</li>
		<li>Select "Analyze>Gels>Label peaks" to obtain a table with the area for each selected peak. These data can be plotted as such (Figure 1D) or can be normalized to the value of a chosen band. This normalization is especially important when pooling values from several replicate gels to determine statistical significance of the results.</li>
</ul>
</li>
</ol>
4. Representative Results
We show a gel with 11 wells loaded with different cell culture supernatants containing different amounts of MMP-2 (Figure 1A). Direct observation of this gel shows obvious differences in MMP-2 concentrations between some of the wells. For example, it is clear that wells # 4 and 5 contain much more MMP-2 than well # 11 or even well # 10. For objective quantification of bands we have used densitometry with the ImageJ software (Figure 1B-D), confirming an approximately 4-fold difference in MMP-2 amounts between well # 11 and wells # 4 and 5 and an approximately 2-fold difference in MMP-2 amounts between well # 10 and wells # 4 and 5.
<img src="/files/ftp_upload/2445/2445fig1.jpg" alt="Figure 1" /> Figure 1. Detection of MMP-2 by gelatin zymography. A, Scanned image of a gelatin gel. Well numbers are indicated at the top of the gel. B, Same gel as in A shown in black and white for densitometry. Each band was selected with a rectangle in ImageJ. C, Two examples of densitometry profiles for the gel shown in A and B (bands # 4 and # 11). A straight line was drawn at the base of each peak to create a closed area. D, Plot of the peak areas for the gel shown in A and B before (left) and after (right) normalization to the density of band # 1.
<img src="/files/ftp_upload/2445/2445fig2.jpg" alt="Figure 2" /> Figure 2. This figure demonstrates how zymography can be used as a semi-quantitative technique. We have loaded serial dilutions (steps of 1:1 dilutions) in 7 consecutive wells and measured the band densities for both pro-MMP-2 and MMP-2.

<ol>
	<li>Luo, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+matrix+metalloproteinases+in+the+morphogenesis+of+the+cerebellar+cortex.">The role of matrix metalloproteinases in the morphogenesis of the cerebellar cortex.</a> Cerebellum. 4, 239-245 (2005).</li><li>Pasternak, B., Aspenberg, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metalloproteinases+and+their+inhibitors+-+diagnostic+and+therapeutic+opportunities+in+orthopedics.">Metalloproteinases and their inhibitors - diagnostic and therapeutic opportunities in orthopedics.</a> Acta Orthop. 80, 693-703 (2009).</li><li>Kessenbrock, K., Plaks, V., Werb, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+metalloproteinases:+regulators+of+the+tumor+microenvironment.">Matrix metalloproteinases: regulators of the tumor microenvironment.</a> Cell. 141, 52-67 (2010).</li><li>Lagente, V., Boichot, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+matrix+metallopreoteinases+in+the+inflammatory+process+of+respiratory+diseases.">Role of matrix metallopreoteinases in the inflammatory process of respiratory diseases.</a> J. Mol. Cell. Cardiol. 48, 440-444 (2010).</li><li>Rodr&#237;guez, D., Morrison, C. J., Overall, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+metallopreoteinases:+what+do+they+not+do?+New+substrates+and+biological+roles+identified+by+murine+models+and+proteomics.">Matrix metallopreoteinases: what do they not do? New substrates and biological roles identified by murine models and proteomics.</a> Biochim. Biophys. Acta. 1803, 39-54 (2010).</li><li>Bourboulia, D., Stetler-Stevenson, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+metalloproteinases+(MMPs)+and+tissue+inhibitors+of+metalloproteinases+(TIMPs):+positive+and+negative+regulators+in+tumor+cell+adhesion.">Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs): positive and negative regulators in tumor cell adhesion.</a> Semin. Cancer Biol. , (2010).</li><li>Heussen, C., Dowdle, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+analysis+of+plasminogen+activators+in+polyacrylamide+gels+containing+sodium+dodecyl+sulfate+and+copolymerized+substrates.">Electrophoretic analysis of plasminogen activators in polyacrylamide gels containing sodium dodecyl sulfate and copolymerized substrates.</a> Analytical Biochem. , 102-196 (1980).</li><li>Lombard, C., Saulnier, J., Wallach, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assays+of+matrix+metalloproteinases+(MMPs)+activities:+a+review.">Assays of matrix metalloproteinases (MMPs) activities: a review.</a> Biochimie. 87, 265-272 (2005).</li><li>Snoek-van Beurden, P. A., Von den Hoff, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zymographic+techniques+for+the+analysis+of+matrix+metalloproteinases+and+their+inhibitors.">Zymographic techniques for the analysis of matrix metalloproteinases and their inhibitors.</a> Biotechniques. 38, 73-83 (2005).</li><li>Kupai, K., Szucs, G., Cseh, S., Hajdu, I., Csonka, C., Csont, T., Ferdinandy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+metalloproteinase+activity+assays:+importance+of+zymography.">Matrix metalloproteinase activity assays: importance of zymography.</a> J. Pharmacol. Toxicol. Methods. 61, 205-209 (2010).</li><li>Kleiner, D. E., Stetler-Stevenson, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+zymography:+detection+of+pictogram+quantities+of+gelatinases.">Quantitative zymography: detection of pictogram quantities of gelatinases.</a> Anal. Biochem. 218, 325-329 (1994).</li><li>Yu, W. H., Woessner, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heparin-enhanced+zymographic+detection+of+matrilysin+and+collagenases.">Heparin-enhanced zymographic detection of matrilysin and collagenases.</a> Anal. Biochem. 293, 38-42 (2001).</li></ol>

No conflicts of interest declared.

We have demonstrated how to perform zymographic analysis of MMP-2 in cell culture supernatants.
The amount of sample to load on a gel must be determined empirically depending on the origin and MMP of interest. Loading too little will prevent detection while loading too much may lead to saturation as there is only so much substrate a protease can digest in the area of the band. If necessary, the sample can be diluted in deionized water prior to mixing with loading buffer or the developing time can de reduced from overnight to 4 hours. It is also possible to concentrate proteins in a solution using concentrators (e.g. Millipore catalog # UFC803024). If bands remain barely visible, it may be necessary to develop the gels for a longer period of time, even up to 48 hours. When preparing samples from tissue culture supernatants, it should be noted that fetal bovine serum (and other sera) contains MMPs that can affect the results. For this reason, we collect all of our supernatants after culture in serum-free medium.
The washes described in paragraphs 2.9 and 2.10 of the protocol are necessary to remove the background staining of digested bands. Longer wash times will remove more background, but will also dim the staining of the substrate throughout the gel. If longer wash times are necessary, we recommend scanning the gel every few hours to select the scan with the best contrast.
This technique can be used to detect other MMPs. For example, MMP-9 can be detected on gelatin gels, and also to a lower extent MMP-1, MMP-8, and MMP-13 as gelatin is not their preferred substrate. MMP-1 and MMP-13 are best detected on collagen zymography while casein is the preferred substrate for MMP-11 and also allows for the detection of MMP-1, MMP-3, MMP-7, MMP-12, and MMP-139. 
MMP-7 (matrilysin) and collagenases (MMP-1 and MMP-13) are difficult to detect when present at low levels in casein or gelatin gels. Addition of heparin to the sample at time of gel loading significantly improves the detection limit for MMP-7. For MMP-1 and MMP-13, the heparin needs to be added to the gel after the electrophoretic run is already under way12.
Since zymography measures enzymatic activity after denaturation and renaturation of the enzymes, it will measure the activity of all MMPs present in the sample. This include enzymes, pro-enzymes, and enzymes bound to endogenous inhibitors (e.g. TIMP-2). It is however possible to determine the level of MMP activation in the sample by comparing the band density of the active enzyme and of that of the pro-enzyme, which will have a slightly higher molecular weight.
Zymography is often not sufficient to identify an MMP. Comparison of the migration level of an MMP with known molecular weight standards does help in the identification but it should be noted that some of these standards contain a reducing agent and that when used under non-reducing conditions they may indicate different molecular weights9. An option is to load a soluble recombinant MMP on the same gel as the test samples. MMPs may however be associated with other proteins, inducing a change in apparent molecular weight. Selective MMP inhibitors can be added to the gel (or part of a gel cut in half) during the incubation in developing buffer as pharmacological tools to identity the MMP of interest.A second technique (Western Blot, immunocytochemistry, or ELISA) is recommended to help in the identification of the MMP of interest. It should be noted that detection limits for Western Blots are often much lower than those of zymographic gels, potentially leading to false-negative results when using this technique.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Xcell SureLock Mini-Cell CE mark electrophoresis apparatus</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>EI0001</td>
<td>&nbsp;</td>
<tr>
<td>Power supply (model 302)</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>93000-744</td>
<td>&nbsp;</td>
<tr>
<td>Novex 10% gelatin zymogram gels, 1.0 mm, 12 wells</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>EC61752BOX</td>
<td>&nbsp;</td>
<tr>
<td>Blue Juice gel loading buffer</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>10816015</td>
<td>&nbsp;</td>
<tr>
<td>Gel loading tips</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>53509-015</td>
<td>&nbsp;</td>
<tr>
<td>Protein molecular weight standard</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC5800</td>
<td>&nbsp;</td>
<tr>
<td>Novex Tris-Glycine-SDS running buffer</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC2675</td>
<td>&nbsp;</td>
<tr>
<td>Novex zymogram renaturing buffer</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC2670</td>
<td>&nbsp;</td>
<tr>
<td>Novex zymogram developing buffer</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC2671</td>
<td>&nbsp;</td>
<tr>
<td>SimplyBlue SafeStain</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>LC6060</td>
<td>&nbsp;</td>
<tr>
<td>Epson Perfection 4490 Photo Scanner</td>
<td>&nbsp;</td>
<td>Amazon</td>
<td>n/a</td>
<td>&nbsp;</td>
<tr>
<td>ImageJ software</td>
<td>&nbsp;</td>
<td>http://rsbweb.nih.gov/ij/ </td>
<td>n/a</td>
<td>Authored by W. Rasband, NIH/NIMH</td>
</tr>		</tbody>
		</table>
		</div>

detection of functional matrix metalloproteinases by zymography

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been identified and are separated into six groups based on their structure and substrate specificity (collagenases, gelatinases, membrane type [MT-MMP], stromelysins, matrilysins, and others). MMPs play a critical role in cell invasion, cartilage degradation, tissue remodeling, wound healing, and embryogenesis. They therefore participate in both normal processes and in the pathogenesis of many diseases, such as rheumatoid arthritis, cancer, or chronic obstructive pulmonary disease1-6. Here, we will focus on MMP-2 (gelatinase A, type IV collagenase), a widely expressed MMP. We will demonstrate how to detect MMP-2 in cell culture supernatants by zymography, a commonly used, simple, and yet very sensitive technique first described in 1980 by C. Heussen and E.B. Dowdle7-10. This technique is semi-quantitative, it can therefore be used to determine MMP levels in test samples when known concentrations of recombinant MMP are loaded on the same gel11.
Solutions containing MMPs (e.g. cell culture supernatants, urine, or serum) are loaded onto a polyacrylamide gel containing sodium dodecyl sulfate (SDS; to linearize the proteins) and gelatin (substrate for MMP-2). The sample buffer is designed to increase sample viscosity (to facilitate gel loading), provide a tracking dye (bromophenol blue; to monitor sample migration), provide denaturing molecules (to linearize proteins), and control the pH of the sample. Proteins are then allowed to migrate under an electric current in a running buffer designed to provide a constant migration rate. The distance of migration is inversely correlated with the molecular weight of the protein (small proteins move faster through the gel than large proteins do and therefore migrate further down the gel). After migration, the gel is placed in a renaturing buffer to allow proteins to regain their tertiary structure, necessary for enzymatic activity. The gel is then placed in a developing buffer designed to allow the protease to digest its substrate. The developing buffer also contains p-aminophenylmercuric acetate (APMA) to activate the non-proteolytic pro-MMPs into active MMPs. The next step consists of staining the substrate (gelatin in our example). After washing the excess dye off the gel, areas of protease digestion appear as clear bands. The clearer the band, the more concentrated the protease it contains. Band staining intensity can then be determined by densitometry, using a software such as ImageJ, allowing for sample comparison.

Watch this Scientific Journal Video about Detection of Functional Matrix Metalloproteinases by Zymography at JoVE.com

Detection of Functional Matrix Metalloproteinases by Zymography

This protocol describes an activity-based assay for detecting matrix metalloproteinases in culture supernatants or body fluids.

Matrix metalloproteinases (MMPs) are zinc-containing endopeptidases. They degrade proteins by cleavage of peptide bonds. More than twenty MMPs have been ...

detection-of-functional-matrix-metalloproteinases-by-zymography

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

83.6K Views.  Baylor College of Medicine. This protocol describes an activity-based assay for detecting matrix metalloproteinases in culture supernatants or body fluids. 

Video: Detection of Functional Matrix Metalloproteinases by Zymography

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo labeling of stem cells with a fluorescent dye, subsequent intravenous injection of the labeled cells and visualization of their accumulation in specific target organs or pathologies. The presented technique demonstrates how we label human mesenchymal stem cells (hMSC) by simple incubation with the lipophilic fluorescent dye DiD (C67H103CIN2O3S) and how we label human embryonic stem cells (hESC) with the FDA approved fluorescent dye Indocyanine Green (ICG). The uptake mechanism is via adherence and diffusion of the lypophilic dye across the phospholipid cell membrane bilayer. The labeling efficiency is usually improved if the cells are incubated with the dye in serum-free media as opposed to incubation in serum-containing media. Furthermore, the addition of the transfection agent Protamine Sulfate significantly improves contrast agent uptake.

This video shows techniques for labeling of human embryonic stem cells and mesenchymal stem cells with fluorescent dyes. This technique can be used for an in vivo tracking of transplanted stem cells with optical imaging and for histopathological correlations with fluorescence microscopy.

Optical imaging (OI) is an easy, fast and inexpensive tool for in vivo monitoring of new stem cell based therapies. The technique is based on ex vivo ...

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 base pairs of DNA associated with two each of histones H2A, H2B, H3, and H41. The N-terminal tails of histones are rich in lysine and arginine and are modified post-transcriptionally by acetylation, methylation, and other post-translational modifications (PTMs). The PTM configuration of nucleosomes can affect the transcriptional activity of associated DNA, thus providing a mode of gene regulation that is epigenetic in nature 2,3. We developed a method called nucleosome ELISA (NU-ELISA) to quantitatively determine global PTM signatures of nucleosomes extracted from cells. NU-ELISA is more sensitive and quantitative than western blotting, and is useful to interrogate the epiproteomic state of specific cell types. This video journal article shows detailed procedures to perform NU-ELISA analysis.

Nucleosome ELISA (NU-ELISA) is a sensitive and quantitative method to detect global patterns of post-translational modifications in preparations of native, intact nucleosomes. These modifications include methylations, acetylations, and phosphorylations at specific histone amino acid residues, and hence NU-ELISA provides a global proteomic assay of the overall chromatin modification states of specific cell types.

The genome of eukaryotes exists as chromatin which contains both DNA and proteins. The fundamental unit of chromatin is the nucleosome, which contains 146 ...

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

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

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

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

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

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

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

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

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Improving the Success Rate of Protein Crystallization by Random Microseed Matrix Screening

Random microseed matrix screening (rMMS) is a protein crystallization technique in which seed crystals are added to random screens. By increasing the likelihood that crystals will grow in the metastable zone of a protein's phase diagram, extra crystallization leads are often obtained, the quality of crystals produced may be increased, and a good supply of crystals for data collection and soaking experiments is provided. Here we describe a general method for rMMS that may be applied to either sitting drop or hanging drop vapor diffusion experiments, established either by hand or using liquid handling robotics, in 96-well or 24-well tray format.

Here we describe a general method for random microseed matrix screening. This technique is shown to significantly increase the success rate of protein crystallization screening experiments, reduce the need for optimization, and provide a reliable supply of crystals for data collection and ligand-soaking experiments.

Random microseed matrix screening  (rMMS) is a protein crystallization technique in which seed crystals are added  to random screens. By increasing the ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

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) ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression regulation at posttranscriptional levels by affecting the stability and translational efficiency of target mRNAs. RNA pull-down technique has been widely used to study RNA-protein interaction, which is necessary to elucidate the mechanism underlying RBPs&#39; as well as long non-coding RNAs&#39; (lncRNAs) function. However, the high lipid abundance in adipocytes poses a technical challenge in conducting this experiment. Here a detailed RNA pull-down protocol is optimized for primary adipocyte culture. An RNA fragment from androgen receptor&#39;s (AR) 3&#39; untranslated region (3&#39;UTR) containing an adenylate-uridylate-rich elementwas used as an example to demonstrate how to retrieve its RBP partner, HuR protein, from adipocyte lystate. The method described here can be applied to detect the interactions between RBPs and noncoding RNAs, as well as between RBPs and coding RNAs.

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression ...

Detection of Functional Matrix Metalloproteinases by Zymography

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Chapters in this video

Labeling Stem Cells with Fluorescent Dyes for non-invasive Detection with Optical Imaging

Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA

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

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

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Improving the Success Rate of Protein Crystallization by Random Microseed Matrix Screening

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

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

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture