Funding provided by The Ohio State University College of Engineering, Biomedical Engineering Department, and the Comprehensive Cancer Center - Arthur G. James Cancer Hospital and Richard J. Solove Research Institute.

1. Preparation of the Resolving Gel Layer
<ol>
	<li>Prepare a 10% polyacrylamide resolving gel solution as per Table 1. Add the Tetramethylethylenediamine (TEMED) and Ammonium Persulfate (APS) immediately prior to pouring the gel as their addition initiates the polymerization reaction.</li>
	<li>Fill an empty 1.5 mm mini-gel cassette half way (5 mL) with the 10% resolving gel solution.</li>
	<li>Add a thin layer of isopropanol (~500 &#181;L) to the top of the polyacrylamide gel to produce a level gel a...

<ol>
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K. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+Aspartic+Proteinase+Activities+Using+Gel+Zymography.">Detection of Aspartic Proteinase Activities Using Gel Zymography.</a> Zymography. , 43-52 (2017).</li><li>van Beurden, P. A. M. S. n. o. e. k. -., Vonden 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 (1), 73-83 (2005).</li><li>Oliver, G. W., Stetler-Stevenson, W. G., Kleiner, D. E., Zymography, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zymography,+Casein+Zymography,+and+Reverse+Zymography:+Activity+Assays+for+Proteases+and+their+Inhibitors.">Zymography, Casein Zymography, and Reverse Zymography: Activity Assays for Proteases and their Inhibitors.</a> Proteolytic Enzymes. Springer Lab Manual. , 63-76 (1999).</li><li>Deshmukh, A. A., Weist, J. L., Leight, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+proteolytic+activity+by+covalent+tethering+of+fluorogenic+substrates+in+zymogram+gels.">Detection of proteolytic activity by covalent tethering of fluorogenic substrates in zymogram gels.</a> BioTechniques. 64 (5), 203-210 (2018).</li><li>Yasothornsrikul, S., Hook, V. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+proteolytic+activity+by+fluorescent+zymogram+in-gel+assays.">Detection of proteolytic activity by fluorescent zymogram in-gel assays.</a> BioTechniques. 28 (6), 1172-1173 (2000).</li><li>Leight, J. L., Alge, D. L., Maier, A. J., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+measurement+of+matrix+metalloproteinase+activity+in+3D+cellular+microenvironments+using+a+fluorogenic+peptide+substrate.">Direct measurement of matrix metalloproteinase activity in 3D cellular microenvironments using a fluorogenic peptide substrate.</a> Biomaterials. 34 (30), 7344-7352 (2013).</li><li>Leight, J. L., Tokuda, E. Y., Jones, C. E., Lin, A. J., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifunctional+bioscaffolds+for+3D+culture+of+melanoma+cells+reveal+increased+MMP+activity+and+migration+with+BRAF+kinase+inhibition.">Multifunctional bioscaffolds for 3D culture of melanoma cells reveal increased MMP activity and migration with BRAF kinase inhibition.</a> Proceedings of the National Academy of Sciences of the United States of America. 112 (17), 5366-5371 (2015).</li><li>Ren, Z., Chen, J., Khalil, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zymography+as+a+Research+Tool+in+the+Study+of+Matrix+Metalloproteinase+Inhibitors.">Zymography as a Research Tool in the Study of Matrix Metalloproteinase Inhibitors.</a> Methods in Molecular Biology. 1626, 79-102 (2017).</li><li>Nagase, H., Fields, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+matrix+metalloproteinase+specificity+studies+using+collagen+sequence-based+synthetic+peptide.">Human matrix metalloproteinase specificity studies using collagen sequence-based synthetic peptide.</a> Biopolymers. 40 (4), 399-416 (1996).</li><li>Mucha, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+Type-1+Matrix+Metalloprotease+and+Stromelysin-3+Cleave+More+Efficiently+Synthetic+Substrates+Containing+Unusual+Amino+Acids+in+Their+P1&#8242;+Positions.">Membrane Type-1 Matrix Metalloprotease and Stromelysin-3 Cleave More Efficiently Synthetic Substrates Containing Unusual Amino Acids in Their P1&#8242; Positions.</a> Journal of Biological Chemistry. 273 (5), 2763-2768 (1998).</li><li>Thimon, V., Belghazi, M., Labas, V., Dacheux, J. -. L., Gatti, J. -. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-+and+two-dimensional+SDS-PAGE+zymography+with+quenched+fluorogenic+substrates+provides+identification+of+biological+fluid+proteases+by+direct+mass+spectrometry.">One- and two-dimensional SDS-PAGE zymography with quenched fluorogenic substrates provides identification of biological fluid proteases by direct mass spectrometry.</a> Analytical Biochemistry. 375 (2), 382-384 (2008).</li><li>Sun, X., Salih, E., Oppenheim, F. G., Helmerhorst, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-based+mass+spectrometric+characterization+of+proteases+and+inhibitors+in+human+saliva.">Activity-based mass spectrometric characterization of proteases and inhibitors in human saliva.</a> Proteomics. Clinical Applications. 3 (7), 810-820 (2009).</li><li>Yu, W., 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> Analytical Biochemistry. 293 (1), 38-42 (2001).</li></ol>

Current zymographic techniques rely on the incorporation of native substrates into polyacrylamide gels for the detection of proteolysis. While these techniques have garnered widespread use, they are still limited in the number of proteases they can detect. Here, a protocol was described in which fluorescent, protease-degradable peptides are incorporated into the polyacrylamide resolving gel. Covalent coupling using an azido-PEG3-maleimide linker molecule enables the separation and detection of a wider variety of protease...

Gel zymography is a biological technique used to measure proteolytic activity within biological samples, such as body fluids or cell culture media1,2,3. The samples are separated by their molecular weights with electrophoresis through a polyacrylamide gel embedded with a degradable substrate. Common degradable substrates include gelatin, casein, collagen and elastin, which have been used to measure the activity of matrix metalloproteinases (MMPs) -1, -2, -3, -7, -8, -9, and -11, in addition to a variety of cathepsins1,2,4,5,6,7,8. After electrophoresis, the enzymes are renatured and allowed to degrade the protein within the gel. In traditional gel zymography, the gel is stained with a protein dye, such as Coomassie&#160;Blue, and protease activity is detected as a loss of signal, i.e., white bands (degradation of protein) on a dark blue background.
Here, we describe a protocol for an alternative method of gel zymography, in which the degradable substrate is a short, fluorogenic peptide covalently incorporated into the polyacrylamide gel (Figure 1). The substitution of synthetic peptides as the degradable substrates enables detection of a wider range of proteases as compared to traditional gel zymography with native proteins9. Covalent linkage of the fluorogenic peptide prevents peptide diffusion and migration during gel electrophoresis observed with previous methods9,10. Furthermore, the use of a fluorogenic substrate enables direct detection of protease activity without additional staining and de-staining steps. The overall goal of this method is the detection of protease activity in biological samples via the covalent incorporation of fluorogenic peptides in zymogram gels.

<table>
	<tbody>
		<tr>
			<td>1.5 mm Empty Gel Cassettes</td>
			<td>ThermoFisher Scientific</td>
			<td>NC2015</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mm, 10 well Empty Gel Cassette Combs</td>
			<td>ThermoFisher Scientific</td>
			<td>NC3510</td>
			<td></td>
		</tr>
		<tr>
			<td>1x Phosphate Buffered Saline</td>
			<td>Fisher Scientific</td>
			<td>10-010-049</td>
			<td></td>
		</tr>
		<tr>
			<td>20% SDS Solution</td>
			<td>Ambion</td>
			<td>AM9820</td>
			<td></td>
		</tr>
		<tr>
			<td>3x Zymography Sample Buffer</td>
			<td>Bio-Rad</td>
			<td>1610764</td>
			<td></td>
		</tr>
		<tr>
			<td>40% (w/v) Acrylamide/Bis (19:1)</td>
			<td>Ambion</td>
			<td>AM9022</td>
			<td></td>
		</tr>
		<tr>
			<td>6 Well Tissue Culture Plates</td>
			<td>ThermoFisher Scientific</td>
			<td>087721B</td>
			<td></td>
		</tr>
		<tr>
			<td>Amicon Ultra-2 Centrifugal Filter Unit (10 kDa MWCO)</td>
			<td>Sigma-Aldrich</td>
			<td>UFC201024</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammounium Persulfate</td>
			<td>Sigma-Aldrich</td>
			<td>A3678</td>
			<td></td>
		</tr>
		<tr>
			<td>Azido-PEG3-Maleimide Kit</td>
			<td>Click Chemistry Tools</td>
			<td>AZ107</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>ThermoFisher Scientific</td>
			<td>BP510100</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Fisher Scientific</td>
			<td>BP231</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Fisher Scientific</td>
			<td>A416P</td>
			<td></td>
		</tr>
		<tr>
			<td>Micro BCA Protein Assay Kit</td>
			<td>ThermoFisher Scientific</td>
			<td>23235</td>
			<td></td>
		</tr>
		<tr>
			<td>N N N&#39; N&#39;-Tetramethylethylenediamine (TEMED)</td>
			<td>Sigma-Aldrich</td>
			<td>T9281</td>
			<td></td>
		</tr>
		<tr>
			<td>PowerPac Basic Power Supply</td>
			<td>Bio-Rad</td>
			<td>1645050</td>
			<td></td>
		</tr>
		<tr>
			<td>Precision Plus Protein Dual Color Standard</td>
			<td>Bio-Rad</td>
			<td>161-0374</td>
			<td></td>
		</tr>
		<tr>
			<td>PrecisionGlide Hypodermic Needles</td>
			<td>Fisher Scientific</td>
			<td>14-826</td>
			<td></td>
		</tr>
		<tr>
			<td>Round Bottom Flask (100 mL)</td>
			<td>Fisher Scientific</td>
			<td>50-873-144</td>
			<td></td>
		</tr>
		<tr>
			<td>Septum Rubber Stopper</td>
			<td>Fisher Scientific</td>
			<td>50-872-546</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Slip Tip Syringe (1 mL)</td>
			<td>Fisher Scientific</td>
			<td>14-823-434</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma hydrochlroide</td>
			<td>Sigma-Aldrich</td>
			<td>T5941</td>
			<td></td>
		</tr>
		<tr>
			<td>Typhoon 9410 Molecular Imager</td>
			<td>GE Amersham</td>
			<td>8149-30-9410</td>
			<td></td>
		</tr>
		<tr>
			<td>Zinc Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>208086</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of protease activity by fluorescent peptide zymography

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using electrophoresis through a resolving gel embedded with a degradable substrate. This method differs from traditional gel zymography in that a quenched fluorogenic peptide is covalently incorporated into the resolving gel instead of full length proteins, such as gelatin or casein. Use of the fluorogenic peptides enables direct detection of proteolytic activity without additional staining steps. Enzymes within the biological samples cleave the quenched fluorogenic peptide, resulting in an increase in fluorescence. The fluorescent signal in the gels is then imaged with a standard fluorescent gel scanner and quantified using densitometry. The use of peptides as the degradable substrate greatly expands the possible proteases detectable with zymographic techniques.

Using the method described here, two fluorescent protease-degradable peptides were incorporated into polyacrylamide gels: GGPQG&#8595;IWGQK(PEG)2C (abbreviated as QGIW throughout the text and figures) and GPLA&#8595;CpMeOBzlWARK(PEG)2C (abbreviated as LACW throughout the text and figures). &#8595; indicates the site of cleavage. QGIW is a collagen-I derived sequence designed to detect cellular collagenases14. LACW is a sequence that...

Watch this Scientific Journal Video about Detection of Protease Activity by Fluorescent Peptide Zymography at JoVE.com

Detection of Protease Activity by Fluorescent Peptide Zymography

Here, we present a detailed protocol for a modified zymographic technique in which fluorescent peptides are used as the degradable substrate in place of native proteins. Electrophoresis of biological samples in fluorescent peptide zymograms enables detection of a wider range of proteases than previous zymographic techniques.

The purpose of this method is to measure the proteolytic activity of complex biological samples. The samples are separated by molecular weight using ...

Current zymographic techniques detect a limited number of proteases. Fluorescent peptide zymography uses fluorescent peptides as the degradable substrate enabling measurement of a greater range of proteases. The main advantage of this method is it's modular design.The sequence of the fluorescent peptide determines which proteases are detected and the fluorescent peptide can easily be swapped with a peptide of a different sequence, an ability to detect other proteases. This technique can help inform the design of peptides'use as degradable cross-linkers in engineered biomaterials such as those used for drug delivery or tissue engineering applications. Incorporation of this peptide into this technique can identify tissue secreted proteases that are responsible for biomaterial degradation.Demonstration of this technique is important in order to visualize the multi-layer approach that is unique compared to other zymography techniques. Demonstrating this technique will be, Ameya Deshmukh, a graduate student from my laboratory. To begin, prepare a 10%polyacrylamide resolving gel solution as outlined in table one of the text protocol.Immediately prior to pouring the gel, add the TEMED and APS. Then, fill and empty 1.5 millimeter mini gel cassette halfway with the resolving gel solution. Add a thin layer of isopropanol to the top of the polyacrylamide gel to produce a level gel and prevent bubbles.Use the leftover polyacrylamide solution to track the progress of the polymerization reaction. When the polyacrylamide in the tube has completely solidified the reaction is complete. While the first resolving gel layer is polymerizing, retrieve the Azido-PEG3-Maleimide kit from storage at 20 degrees Celsius and allow the components to reach room temperature.Then, dissolve the components of vial two in the manufacturer recommended volume of DMSO. And vortex for 30 seconds to ensure the liquids are well-mixed. Transfer the contents of vial one into a clean, dry 100 millileter round bottom flask that contains a stir bar.Immediately insert a rubber septum stopper with a diaphragm that can be punctured with a syringe into the mouth of the flask. Next, insert two 18-gauge syringe needles into the diaphragm. Connect one of the syringe needles to an inert gas tank and allow the gas to fill the round bottom flask for three minutes.Then, shut off the inert gas and detach it from the needle. Using a syringe, inject the full contents of vial two into the flask. Remove both of the needles and the syringe.Allow the components to mix for 30 minutes at room temperature while stirring. After this remove the rubber septum stopper and transfer the contents to a clean five milliliter centrifuge tube. Once the first resolving gel layer has polymerized pour off the isopropanol layer.Pipet one milliliter of de-ionized water on top of the gel and then pour the water off to rinse the gel. Retrieve the thiol functionalized fluorescent peptide from storage at 80 degrees Celsius. Allow it to thaw at room temperature.Prepare a 10%resolving gel solution containing the Azido-PEG3-Maleimide linker molecule and the fluorescent peptide as outlined in table one of the text protocol. Immediately prior to pouring the gel add the TMED and APS. Next, fill half of the remaining portion of the gel cassette with the peptide resolving gel solution.Add a thin layer of isopropanol to the top of the polyacrylamide gel to produce a level gel and prevent bubbles. Use the leftover polyacrylamide solution to track the progress of the polymerization reaction. After the reaction is complete, pour off the isopropanol layer.Pipet one milliliter of de-ionized water on top of the gel and then pour the water off to rinse the gel. If not using the gels immediately, store them by immersing them in a plastic box filled with 100 milliliters of 1x PBS at four degrees Celsius. Wrap the box in aluminum foil to prevent photobleaching.First, prepare a 5%stacking gel solution as outlined in table one of the text protocol. Immediately prior to pouring the gel add the TMED and APS. Fill the remaining empty portion of the gel cassette with the stacking gel solution.Quickly insert a 1.5 millimeter gel comb into the stacking gel layer making sure no bubbles remain trapped under the wells. Use the leftover polyacrylamide solution to track the progress of the polymerization reaction. When the reaction is complete, gently remove the comb and the tape from the back of the gel cassette.To begin, dissolve samples in conventional zymography sample buffer. Add 400 milliliter os 1X tris glycene SDS running buffer to the gel apparatus. Load up to 35 microliters of sample per well.Run the samples at 120 volts at four degrees Celsius for one and a half hours or until the molecular weight standards indicate that the proteases of interest are within the peptide-resolving gel layer. After this, remove the gels from the plastic cassette. Wash the gels three times in re-naturing buffer at room temperature under gentle agitation.With each wash, lasting 10 minutes. Transfer gels to a developing buffer solution for 15 minutes. Then replace the solution with fresh developing buffer and incubate at 37 degrees Celsius under gentle agitation for 24 hours.After this, use a fluorescent gel scanner imager with the appropriate excitation and emission filters to image the gels. In this study to fluorescent protease degradable peptides are incorporated into polyacrylamide gels. QGIW is a collagen one derived sequence designed to detect cellular collagenesis, while LACW is a sequence that has been optimized for the detection of MMP-14 and MMP-11.Fluorescent imaging reveals numerous bands within the LACW peptide gels, While only a single band is seen in the QGIW gels. When compared to gelatin zymography, LACW gels are able to detect more proteolytic bands, which demonstrates the ability of peptide zymography to detect a wider range of proteases present within biological samples than traditional methods using native substrates. Peptide zymography cells are then incubated in development buffer containing either GM6001, a broad spectrum MMP-inhibitor, or E64, a general cathepsin inhibitor.Treatment of the LACW peptide gels with GM6001, decreases the intensity of the bands. While treatment with E64 has no discernible effect. Treatment of the QGIQ peptide gels with GM6001 results in complete ablation of the previously seen bands.While E64 has no effect. Gelatin zymography is conducted using purified, activated MMP-9 to compare the sensitivity of peptide zymography to the current gold standard. LACW gels are able to detect the smallest concentrations of MMP-9.Proteases require specific conditions to re-nature and become activated. Carefully prepare the buffer solutions to ensure that the composition and PH are correct. This method can be complemented by existing techniques to verify the identity and perform further analysis of the measured proteases.This includes western blotting, real-time PCR, and mass spectrometry. Be cautious when handling polyacrylamide. Unpolymerized solution is considered a potent neurotoxin and appropriate personal protective equipment should always be worn when handling it.

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12.5K visualizações.  Ohio State University. As técnicas zymográficas atuais detectam um número limitado de proteases. A zymografia fluorescente do peptídeo usa peptídeos fluorescentes como o substrato degradável que permite a medição de uma maior gama de proteases. A principal vantagem deste método é seu design modular.A sequência do peptídeo fluorescente determina quais proteases são detectadas e o peptídeo fluorescente pode ser facilmente trocado por um peptídeo de uma sequência diferente, uma capacidade de det...