Name: Detection of Functional Matrix Metalloproteinases by Zymography (Video) | JoVE Journal
Uploaded: 2010-11-08T17:00:00.000+00:00
Duration: 9 min 30 s
Description: Learn gelatin zymography for detecting functional matrix metalloproteinases in biological samples. This method enables quantitative analysis of MMP activity through electrophoresis and band density measurement.

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5 minutes

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°C. The samples must not contain reducing agents (such a β-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 μL 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 μL 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°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&gt;Gels&gt;Select next lane". Repeat until all bands are selected (Figure 1B).</li>
		<li>Press "3" or select "Analyze&gt;Gels&gt;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&gt;Gels&gt;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="Electrophoresis results; gel bands, absorption peaks, bar graph analysis of band intensities." data-altupdatedat="1788235205000" data-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="Gel electrophoresis and bar chart showing protein levels of Pro-MMP-2 and MMP-2 across samples." data-altupdatedat="1788235205000" data-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/pubmed?term=((The+role+of+matrix+metalloproteinases+in+the+morphogenesis+of+the+cerebellar+cortex%5BTitle%5D)+AND+%22Cerebellum%22%5BJournal%5D)">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/pubmed?term=((Metalloproteinases+and+their+inhibitors+-+diagnostic+and+therapeutic+opportunities+in+orthopedics%5BTitle%5D)+AND+%22Acta+Orthop%22%5BJournal%5D)">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/pubmed?term=((Matrix+metalloproteinases%3A+regulators+of+the+tumor+microenvironment%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">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/pubmed?term=((Role+of+matrix+metallopreoteinases+in+the+inflammatory+process+of+respiratory+diseases%5BTitle%5D)+AND+%22J.+Mol.+Cell.+Cardiol%22%5BJournal%5D)">Role of matrix metallopreoteinases in the inflammatory process of respiratory diseases.</a> J. Mol. Cell. Cardiol. 48, 440-444 (2010).</li>
<li>Rodríguez, D., Morrison, C. J., Overall, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Matrix+metallopreoteinases%3A+what+do+they+not+do%3F+New+substrates+and+biological+roles+identified+by+murine+models+and+proteomics%5BTitle%5D)+AND+%22Biochim.+Biophys.+Acta%22%5BJournal%5D)">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/pubmed?term=((Matrix+metalloproteinases+%28MMPs%29+and+tissue+inhibitors+of+metalloproteinases+%28TIMPs%29%3A+positive+and+negative+regulators+in+tumor+cell+adhesion%5BTitle%5D)+AND+%22Semin.+Cancer+Biol%22%5BJournal%5D)">Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs): positive and negative regulators in tumor cell adhesion.</a> Semin. Cancer Biol. , Forthcoming (2010).</li>
<li>Heussen, C., Dowdle, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Electrophoretic+analysis+of+plasminogen+activators+in+polyacrylamide+gels+containing+sodium+dodecyl+sulfate+and+copolymerized+substrates%5BTitle%5D)+AND+%22Analytical+Biochem%22%5BJournal%5D)">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/pubmed?term=((Assays+of+matrix+metalloproteinases+%28MMPs%29+activities%3A+a+review%5BTitle%5D)+AND+%22Biochimie%22%5BJournal%5D)">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/pubmed?term=((Zymographic+techniques+for+the+analysis+of+matrix+metalloproteinases+and+their+inhibitors%5BTitle%5D)+AND+%22Biotechniques%22%5BJournal%5D)">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/pubmed?term=((Matrix+metalloproteinase+activity+assays%3A+importance+of+zymography%5BTitle%5D)+AND+%22J.+Pharmacol.+Toxicol.+Methods%22%5BJournal%5D)">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/pubmed?term=((Quantitative+zymography%3A+detection+of+pictogram+quantities+of+gelatinases%5BTitle%5D)+AND+%22Anal.+Biochem%22%5BJournal%5D)">Quantitative zymography: detection of pictogram quantities of gelatinases.</a> Anal. Biochem. 218, 325-329 (1994).</li>
<li>Yu, W. H., Woessner, J. F. Jr <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Heparin-enhanced+zymographic+detection+of+matrilysin+and+collagenases%5BTitle%5D)+AND+%22Anal.+Biochem%22%5BJournal%5D)">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.

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

detection of functional matrix metalloproteinases by zymography

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.

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 ...

The overall goal of this procedure is to detect functional matrix metalloprotein ais. This is achieved by first preparing the sample for zy by adding a loading buffer free of reducing agents agility. Gelatin, ZY Graham gel is then loaded with the sample and allowed to run until the tracking dye reaches the end of the gel.Following electrophoresis, the gel is re natured, developed, stained and de stain. The final step of the procedure is to scan the gel for data analysis. Ultimately, results can be obtained that show the activity of matrix metalloproteinase in the samples through band density analysis.Hi, I'm from the laboratory of Dr.Kristin bitten in the Department of Molecular Physiology and biophysics at the LER College of Medicine. Today we'll show you a procedure for detecting matrix metallo Persis by phy. We use this procedure in our laboratory to study matrix Metallo Persis secreted by cultured primary cell.So let's get studied. Prior to the start of this protocol, prepare all matrix metalloproteinase or MMPs adequately to maintain the function of the enzymes. The samples can be used immediately or stored at minus 80 degrees Celsius.Here MMP two, also known as gelatinize A or type four collagenase, is used to demonstrate visualization of MP activity with synography. Once MMP enzymes have been prepared, obtain a 10%gelatin zy gram gel pouch containing a gel inside a cassette. Rinse the cassette with deionized water.Remove the protective tape from the bottom of the cassette and the comb from the top of the gel. Rinse the wells three times with tris glycine. SDS running buffer.Place the gel into a mini cell electrophoresis apparatus ensuring that the smaller side of the cassette faces inward. Lock the gel into place with the gel tension wedge. Fill the top chamber of the apparatus with tris glycine SDS running buffer above the level of the wells and check for any leaks.Fill the lower chamber of the gel electrophoresis set up with running buffer as well. Then load 10 microliters of protein molecular marker. Mix an equal amount of the sample to a gel loading buffer, which does not contain reducing agents.Unlike regular SDS page, do not boil the samples and instead directly load them into the wells of the gel. Using gel loading tips, these wells can hold up to 20 microliters of volume. Place the lid on the mini cell and connect the electrode cords to the power supply.Switch the power supply on and set the gel to run at 125 volts constant for 90 minutes. Check the formation of small bubbles on the wire of the lower chamber indicating current circulation when running in the first gel. Monitor the progress of migration every 15 minutes using the bromo phenol blue included in the loading buffer as an indicator for each gel.Prepare 100 milliliters of one time xmo gram Rena buffer, and 200 milliliters of xmo gram developing buffer, both in deionized water. When the bromo phenol 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 a gel knife, cut a corner to mark the direction of the gel. Carefully remove the gel from the cassette and place it into a container with 100 milliliters of Rena Buffer.Incubate the gel for 30 minutes At room temperature with gentle vegetation, remove the Rena buffer and add 100 milliliters of developing buffer to the gel. Incubate for 30 minutes at room temperature with gentle vegetation. Then remove the developing buffer and add an additional 100 milliliters of fresh developing buffer to the gel.Incubate the gel overnight at 37 degrees Celsius. Following overnight incubation, remove the developing buffer and wash the gel by incubating it with deionized water and a gentle vegetation. For five minutes at room temperature, repeat this wash two times.Place the gel on a plastic sheet protector and remove bubbles. Then scan the gel with a photo scanner to save the exact positioning of the protein standard bands as they will become barely visible. After gel staining.Stain the gel by adding 20 milliliters of simply blue safe stain to the gel. Incubate the gel for one hour at room temperature. Under gentle agitation, remove the simply blue safe stain and detain the gel in 100 milliliters of deionized water at room temperature under gentle agitation.After one hour, replace with fresh deionized water and continue to incubate the gel for at least one hour. To analyze the data using cytometry first carefully remove the gel from the water and place it in a plastic sheet protector. Using a photo scanner, scan the gel with a resolution of 300 dots per inch or higher.Save the image in a TIFF format. Open the TIFF file in Image J or similar quantification software to measure band intensities. Visualize the image in black and white by selecting image type eight bit.Use the rectangular selection tool to outline the first band drawing a rectangle that is at least twice higher than it is wide. Select analyze gels. Select first lane, and the band will be selected.Then a new rectangle will appear. Move it to the next band and select Analyze gels, select next lane. Repeat this process until all the bands are selected.Select analyze gels plot lanes to generate the profile plot for each band. Use the straight line selection to draw baselines so that the peak of interest is a completely enclosed area. Select the wand tool and click inside each peak to select it.Select analyze gels label peaks to obtain a table with the area for each selected peak. These data can be plotted as such 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.Each well of the gel shown here was loaded with a different cell culture. Supinate containing different amounts of MMP two. Direct observation of this gel shows obvious differences in MMP two concentrations between some of the wells.For example, it is clear that both wells four and five contain much more MMP two than wells 10 and 11 for objective quantification of bands. Dense cytometry was used with the Image J software. This software revealed an approximate fourfold difference in mmp.Two amounts between, well, 11 and wells four and five. Similarly, a twofold difference in MMP two amounts was observed between wells 10 and wells four and five. We are just showing you how to detect functional metrics.Metal persis by xog. When you do this procedure, it is important to remember not to boil your sample. Otherwise you cannot see any bands on your gel.So that's it. Thanks for watching and good luck with your experiment.

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

Research

JoVE Journal

Biology

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Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

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Accurate mass measurement represents an important step during the investigation of proteins. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) can be used for such determination and its key advantage is the tolerance to salts, detergents and contaminants. Here we illustrate an accessible approach for the analysis of proteins larger than 100 kDa by MALDI-MS.

Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa

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2D and 3D Matrices to Study Linear Invadosome Formation and Activity

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Detection of Protease Activity by Fluorescent Peptide Zymography

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Proteinases are vital for bacterial pathogenesis, enabling invasion and immune evasion. Single-step extraction of cytoplasmic and membrane proteins of bacteria in their active form is harrowing. This video article demonstrates the extraction of whole cellular proteins of Leptospira for zymography analysis to detect and characterize gelatinases.

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Detection of Functional Matrix Metalloproteinases by Zymography

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