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 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 (~40 min).</li>
</ol>
2. Preparation of the Azido-PEG3-maleimide Linker Molecule
<ol>
	<li>While the first resolving gel layer is polymerizing, retrieve the azido-PEG3-maleimide kit from the -20 &#176;C storage and allow the components to reach room temperature. There are two components in each kit. Vial 1 contains a maleimide-NHS ester, an off-white to grey solid. Vial 2 contains azido-PEG3-amine, a slightly yellow oil. 
	NOTE: The authors suggest using the 25 mg azido-PEG3-maleimide kit as it can only be stored for short periods of time (1-2 hours) at -20 &#176;C after being prepared before it begins to degrade. 25 mg is sufficient to produce 10 peptide gels. Use gels within 3 weeks of preparation.</li>
	<li>Dissolve the components of Vial 2 in the manufacturer recommended volume of dimethyl sulfoxide (DMSO) and vortex for 30 s to ensure the liquids have been mixed well.</li>
	<li>Transfer the contents of Vial 1 into a clean, dry 100 mL round-bottom flask containing a stir bar. 
	NOTE: Rinse the flask with acetone and dry completely prior to usage to prevent moisture from interfering with the reaction.</li>
	<li>Immediately insert a rubber septum stopper with a diaphragm that can be punctured with a syringe into the mouth of the flask. Work quickly to prevent moisture from entering the flask.</li>
	<li>Insert two 18 gauge syringe needles into the diaphragm and connect one to an inert gas tank (e.g. argon gas). Allow the inert gas to fill the flask for 3 min. Mix the components of Vials 1 and 2 under inert gas to prevent undesirable reaction products. 
	CAUTION: The second syringe needle is to provide a vent, thereby allowing the atmospheric air contained within the flask to flow out of the flask as it fills with inert gas. Do not forget to include a vent needle!</li>
	<li>Shut off the inert gas and detach it from the needle. Using a syringe, inject the full contents of Vial 2 into the flask.</li>
	<li>Remove both needles and syringe and allow the components to mix for 30 min at room temperature while stirring.</li>
	<li>Remove the rubber septum stopper and transfer the contents to a clean 5 mL centrifuge tube. The azido-PEG3-maleimide solution must be used within 1 hour at room temperature.</li>
</ol>
3. Preparation of the Peptide Resolving Gel Layer
<ol>
	<li>Once the first resolving gel layer has polymerized, pour off the isopropanol layer. Rinse the top of the gel by pipetting 1 mL of deionized water on the top of the gel and then pour off the water.</li>
	<li>Retrieve the thiol-functionalized fluorescent peptide from -80 &#176;C storage and allow it to thaw at room temperature. 
	NOTE: The thiol-functionalized peptide can be prepared as described previously11,12. Commercially available peptides can also be used but require the addition of a terminal cysteine residue to enable the maleimide-thiol click reaction. Dissolve the peptide to a stock concentration of 10 mM and store it at -80 &#176;C in small (30 uL) aliquots to limit repeated freeze-thaw cycles.</li>
	<li>Prepare a 10% resolving gel solution containing the azido-PEG3-maleimide linker molecule and the fluorescent peptide as per Table 1. Add the TEMED and APS immediately prior to pouring the gel as their addition initiates the polymerization reaction.</li>
	<li>Fill half of the remaining portion of the gel cassette (3 mL) with the peptide resolving gel solution. 
	NOTE: A multi-layer resolving gel approach reduces the amount of peptide and linker necessary for each gel. The size of the peptide resolving gel layer can be adjusted to accommodate a larger range of molecular weights as needed.</li>
	<li>Pipette a thin layer of isopropanol (~500 &#181;L) 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 (~40 min). 
	NOTE: The fluorescent peptide is light sensitive. Keep the gels covered with aluminum foil to prevent photobleaching during gel preparation, electrophoresis, washing and development.</li>
	<li>Pour off the isopropanol layer and rinse the top of the peptide resolving gel with deionized water as in step 3.1.</li>
	<li>If using the gels immediately, proceed to step 4, otherwise, immerse the prepared gels in 100 mL of 1x phosphate buffered saline (PBS) at 4 &#176;C in a plastic box to prevent the gels from drying out. Wrap the box in aluminum foil to prevent photobleaching. Gels can be stored in PBS for up to 3 weeks prior to usage.</li>
</ol>
4. Preparation of the Stacking Gel
<ol>
	<li>Prepare a 5% stacking gel solution as per Table 1. Add the TEMED and APS immediately prior to pouring the gel as their addition initiates the polymerization reaction.</li>
	<li>Fill the remaining empty portion of the gel cassette (~2 mL) with the stacking gel solution.</li>
	<li>Quickly insert a 1.5 mm 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 polyacrylamide in the tube has completely solidified, the reaction is complete (~10 min).</li>
	<li>Gently remove the comb and the tape from the back of the gel cassette.</li>
</ol>
5. Preparation of Biological Samples for Electrophoresis
<ol>
	<li>Prepare conditioned cell media, cell lysates, tissue homogenates, and MMP standards as described elsewhere under non-reducing conditions2. Do not heat samples.</li>
	<li>For example, prepare conditioned cell media as follows:
	<ol>
		<li>Plate 40,000 cells/cm2 cells in a 6-well plate in 10% fetal bovine serum (FBS) culture media. Incubate cells in a humidified chamber (5% CO2 at 37 &#176;C) for 24 hours and allow them to reach 70-80% confluence. 
		NOTE: If the cells have not reached the desired confluency after 24 hours, allow them to grow in 10% FBS culture media for an additional 24 hours.</li>
		<li>Wash the cells twice with PBS and add 2 mL of serum-free culture media. Incubate the cells in a humidified chamber (5% CO2 at 37 &#176;C) for an additional 24 hours.</li>
		<li>Using a serological pipette, collect the conditioned media from each well. Centrifuge the media at 1200 rpm for 3 minutes to remove any cell debris. Take the supernatant and concentrate using a 15 mL, 10 kDa molecular weight cutoff centrifugal filter unit. Centrifuge the filter units at 4,000 x g for 15 min in a swinging bucket rotor or 5,000 x g for 15 min in a fixed angle rotor. 
		NOTE: This step is optional but can enhance the intensity of the proteolytic bands in the peptide zymography gels.</li>
		<li>Transfer the concentrated filtrate to a fresh 1.5 mL centrifuge tube. Aliquot and store samples at -80 &#176;C for up to three freeze-thaw cycles.</li>
	</ol>
	</li>
	<li>Quantify protein content using a standard protein quantification assay (e.g. BCA, Bradford Assay, etc.).</li>
</ol>
6. Electrophoresis of Biological Samples in Peptide Zymography Gels
<ol>
	<li>Dissolve samples in conventional zymography sample buffer (62.5 mM Tris-HCl, pH 6.8, 25% glycerol, 4% SDS, 0.01% bromophenol blue). For cell and tissue samples, ~30 &#181;g of total protein per well is recommended, and 50-100 ng of protein for MMP standards.</li>
	<li>Add 400 mL of 1x Tris-Glycine SDS Running Buffer to the gel apparatus. Load up to 35 &#181;L of sample per well.&#160;Run the samples at 120 V at 4 &#176;C for 1.5 hours or until the molecular weight standards indicate that the proteases of interest are within the peptide resolving gel layer (which has a visible orange color). 
	NOTE: Most MMPs and their variants fall within the range of 35-100 kDa. When the molecular weight standards indicate that those weights are within the peptide resolving gel layer, electrophoresis can be stopped. The same principle can be applied to other classes of proteases with known molecular weights. If there is an interest in detecting multiple proteases over a larger range of molecular weights, reduce the size of the resolving gel layer and increase the size of the peptide resolving gel layer.</li>
	<li>Following electrophoresis, remove the gels from the plastic cassette and wash gels three times for 10 min each at room temperature under gentle agitation in renaturing buffer containing 2.5% Triton X-100, 1 &#181;M ZnCl2, and 5 mM CaCl2 in 50 mM Tris-HCl, pH 7.5.</li>
	<li>Transfer gels to a developing buffer solution containing 1% Triton X-100, 1 &#181;M ZnCl2 and 5 mM CaCl2 in 50 mM Tris-HCl, pH 7.5 for 15 min. Replace with fresh developing buffer solution and incubate gels at 37 &#176;C under gentle agitation for 24 hours, making sure the gels are fully submersed in the solution.</li>
</ol>
7. Imaging of Peptide Zymography Gels
<ol>
	<li>After 24 hours, image gels using a fluorescent gel scanner/imager using the appropriate excitation and emission filters. For example, the peptide gels shown in the representative results are conjugated with Fluorescein and were imaged using an excitation filter of 488 nm and an emission filter of 521 nm. Using the appropriate filters for your fluorophore will maximize the detection of proteolytic activity. 
	NOTE: Images can also be taken with a gel imager equipped with a UV transilluminator, often used for the imaging of DNA gels stained with ethidium bromide. Image the gels using the UV transilluminator (365 nm) setting and an emission filter of 590 nm.</li>
	<li>Conduct densitometric evaluation of band intensities using ImageJ as described elsewhere13.</li>
</ol>

<ol><li>Vandooren, J., Geurts, N., Martens, E., Vanden Steen, P. E., Opdenakker, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zymography+methods+for+visualizing+hydrolytic+enzymes%5BTitle%5D)+AND+%22Nature+Methods%22%5BJournal%5D)">Zymography methods for visualizing hydrolytic enzymes.</a> Nature Methods. 10 (3), 211-220 (2013).</li>
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<li>Inanc, S., Keles, D., Oktay, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((An+improved+collagen+zymography+approach+for+evaluating+the+collagenases+MMP-1%2C+MMP-8%2C+and+MMP-13%5BTitle%5D)+AND+%22BioTechniques%22%5BJournal%5D)">An improved collagen zymography approach for evaluating the collagenases MMP-1, MMP-8, and MMP-13.</a> BioTechniques. 63 (4), 174-180 (2017).</li>
<li>Perera, H. K. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detection+of+Aspartic+Proteinase+Activities+Using+Gel+Zymography%5BTitle%5D)+AND+%22Zymography%22%5BJournal%5D)">Detection of Aspartic Proteinase Activities Using Gel Zymography.</a> Zymography. , 43-52 (2017).</li>
<li>van Beurden, P. A. M. S. noek-, Vonden 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 (1), 73-83 (2005).</li>
<li>Oliver, G. W., Stetler-Stevenson, W. G., Kleiner, D. E., Zymography, Zymography, Casein Zymography, and Reverse Zymography: Activity Assays for Proteases and their Inhibitors. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aOliver%2C+GW+%22Proteolytic+Enzymes.+Springer+Lab+Manual%22">Proteolytic Enzymes. Springer Lab Manual</a>. , Springer. Berlin, Heidelberg. 63-76 (1999).</li>
<li>Deshmukh, A. A., Weist, J. L., Leight, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detection+of+proteolytic+activity+by+covalent+tethering+of+fluorogenic+substrates+in+zymogram+gels%5BTitle%5D)+AND+%22BioTechniques%22%5BJournal%5D)">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/pubmed?term=((Detection+of+proteolytic+activity+by+fluorescent+zymogram+in-gel+assays%5BTitle%5D)+AND+%22BioTechniques%22%5BJournal%5D)">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/pubmed?term=((Direct+measurement+of+matrix+metalloproteinase+activity+in+3D+cellular+microenvironments+using+a+fluorogenic+peptide+substrate%5BTitle%5D)+AND+%22Biomaterials%22%5BJournal%5D)">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/pubmed?term=((Multifunctional+bioscaffolds+for+3D+culture+of+melanoma+cells+reveal+increased+MMP+activity+and+migration+with+BRAF+kinase+inhibition%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">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/pubmed?term=((Zymography+as+a+Research+Tool+in+the+Study+of+Matrix+Metalloproteinase+Inhibitors%5BTitle%5D)+AND+%22Methods+in+Molecular+Biology%22%5BJournal%5D)">Zymography as a Research Tool in the Study of Matrix Metalloproteinase Inhibitors.</a> Methods in Molecular Biology. 1626, Clifton, N.J. 79-102 (2017).</li>
<li>Nagase, H., Fields, G. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Human+matrix+metalloproteinase+specificity+studies+using+collagen+sequence-based+synthetic+peptide%5BTitle%5D)+AND+%22Biopolymers%22%5BJournal%5D)">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/pubmed?term=((Membrane+Type-1+Matrix+Metalloprotease+and+Stromelysin-3+Cleave+More+Efficiently+Synthetic+Substrates+Containing+Unusual+Amino+Acids+in+Their+P1%E2%80%B2+Positions%5BTitle%5D)+AND+%22Journal+of+Biological+Chemistry%22%5BJournal%5D)">Membrane Type-1 Matrix Metalloprotease and Stromelysin-3 Cleave More Efficiently Synthetic Substrates Containing Unusual Amino Acids in Their P1′ 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/pubmed?term=((One-+and+two-dimensional+SDS-PAGE+zymography+with+quenched+fluorogenic+substrates+provides+identification+of+biological+fluid+proteases+by+direct+mass+spectrometry%5BTitle%5D)+AND+%22Analytical+Biochemistry%22%5BJournal%5D)">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/pubmed?term=((Activity-based+mass+spectrometric+characterization+of+proteases+and+inhibitors+in+human+saliva%5BTitle%5D)+AND+%22Proteomics.+Clinical+Applications%22%5BJournal%5D)">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/pubmed?term=((Heparin-Enhanced+Zymographic+Detection+of+Matrilysin+and+Collagenases%5BTitle%5D)+AND+%22Analytical+Biochemistry%22%5BJournal%5D)">Heparin-Enhanced Zymographic Detection of Matrilysin and Collagenases.</a> Analytical Biochemistry. 293 (1), 38-42 (2001).</li>
</ol>

The authors have nothing to disclose.

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&#039; N&#039;-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↓IWGQK(PEG)2C (abbreviated as QGIW throughout the text and figures) and GPLA↓CpMeOBzlWARK(PEG)2C (abbreviated as LACW throughout the text and figures). ↓ indicates the site of cleavage. QGIW is a collagen-I derived sequence designed to detect cellular collagenases14. LACW is a sequence that has been op...

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 ...

detection-of-protease-activity-by-fluorescent-peptide-zymography

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Cancer Research

12.6K Views.  Ohio State University. Huidige zymografische technieken detecteren een beperkt aantal proteasen. Fluorescerende peptide zymografie maakt gebruik van fluorescerende peptiden als het afbreekbare substraat dat het meten van een groter bereik van proteasen mogelijk maakt. Het belangrijkste voordeel van deze methode is het modulaire ontwerp.De volgorde van het fluorescerende peptide bepaalt welke proteasen worden gedetecteerd en het fluorescerende peptide kan gemakkelijk worden verwisseld met een peptide van een ...

Video: Detection of Protease Activity by Fluorescent Peptide Zymography

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will survive longer than five years. In order to understand and mimic the microenvironment of pancreatic tumors, we utilized a murine orthotopic model of pancreatic cancer that allows non-invasive imaging of tumor progression in real time. Pancreatic cancer cells expressing green fluorescent protein (PANC-1 GFP) were suspended in basement membrane matrix, high concentration, (e.g., Matrigel HC) with serum-free media and then injected into the tail of the pancreas via laparotomy. The cell suspension in the high concentration basement membrane matrix becomes a gel-like substance once it reaches room temperature; therefore, it gels when it comes in contact with the pancreas, creating a seal at the injection site and preventing any cell leakage. Tumor growth and metastasis to other organs are monitored in live animals by using fluorescence. It is critical to use the appropriate filters for excitation and emission of GFP. The steps for the orthotopic implantation are detailed in this article so researchers can easily replicate the procedure in nude mice. The main steps of this protocol are preparation of the cell suspension, surgical implantation, and whole body fluorescent in vivo imaging. This orthotopic model is designed to investigate the efficacy of novel therapeutics on primary and metastatic tumors.

A procedure to implant green fluorescent protein-expressing pancreatic cancer cells (PANC-1 GFP) orthotopically into the pancreas of Balb-c Ola Hsd-Fox1nu mice to assess tumor progression and metastasis is presented here.

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will ...

In Vitro Imaging and Quantification of the Drug Targeting Efficiency of Fluorescently Labeled GnRH Analogues

GnRH analogues are effective targeting moieties and able to deliver anticancer agents selectively into malignant tumor cells which highly express GnRH receptors. However, the quantitative analysis of GnRH analogues&#39; cellular uptake and the investigated cell types in GnRH-based drug delivery systems are currently limited. Previously introduced, selectively labeled fluorescent GnRH I, -II and -III derivatives provide great detectability, and they have suitable chemical properties for reproducible and robust experiments. We also found that the appropriate up-to-date methods with these labeled GnRH analogues could offer novel information about the GnRH-based drug delivery systems. This manuscript introduces some simple and fast experiments regarding the cellular uptake of [D-Lys6(FITC)]-GnRH-I, [D-Lys6(FITC)]-GnRH-II and [Lys8(FITC)]-GnRH-III on the EBC-1 (lung), the BxPC-3 (pancreas) and on the Detroit-562- (pharynx) malignant tumor cells. In parallel with these GnRH-FITC conjugates, the cell surface level of GnRH-I receptors was also examined on these cell lines before and after the GnRH treatment by confocal laser scanning microscopy. The cellular uptake of GnRH-FITC conjugates was quantified by fluorescence-activated cell sorting. In these experiments minor differences among GnRH analogues and major differences among cell types was observed. The significant differences among cell lines are correlated with their distinct level of cell surface GnRH-I receptors. The introduced experiments contain practical methods to visualize, quantify and compare the uptake efficiency of GnRH-FITC conjugates in a time- and concentration-dependent manner on various adherent cell cultures. These results could predict the drug targeting efficiency of GnRH conjugates on the given cell culture, and offer a good basis for further experiments in the examination of GnRH-based drug delivery systems.

In Vitro Imaging and Quantification of the Drug Targeting Efficiency of Fluorescently Labeled GnRH Analogues

Selectively labeled fluorescent GnRH-I, -II and -III derivatives are reliable tools for tracking and quantifying their cellular uptake. This manuscript introduces experiments to visualize, quantify and compare the uptake efficiency of these GnRH conjugates in various cell lines.

GnRH analogues are effective targeting moieties and able to deliver anticancer agents selectively into malignant tumor cells which highly express GnRH ...

Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, they are important drug targets. We have developed a magnetic-agarose-bead-based assay platform for the investigation of proteolytic activity, which is based on the use of recombinant fusion protein substrates. In order to demonstrate the use of this assay system, a protocol is presented on the example of human immunodeficiency virus type 1 (HIV-1) protease. The introduced assay platform can be utilized efficiently in the biochemical characterization of proteases, including enzyme activity measurements in mutagenesis, kinetic, inhibition, or specificity studies, and it may be suitable for high-throughput substrate screening or may be adapted to other proteolytic enzymes.
In this assay system, the applied substrates contain N-terminal hexahistidine (His6) and maltose-binding protein (MBP) tags, cleavage sites for tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein. The substrates can be efficiently produced in Escherichia coli cells and easily purified using nickel (Ni)-chelate-coated beads. During the assay, the proteolytic cleavage of bead-attached substrates leads to the release of fluorescent cleavage fragments, which can be measured by fluorimetry. Additionally, cleavage reactions can be analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A protocol for the in-gel renaturation of assay components is also described, as partial renaturation of fluorescent proteins enables their detection based on molecular weight and fluorescence.

Here, we present the detailed procedure of a recently developed protease assay platform utilizing N-terminal hexahistidine/maltose-binding protein and fluorescent protein-fused recombinant substrates attached to the surface of nickel-nitrilotriacetic acid magnetic agarose beads. A subsequent in-gel analysis of the assay samples separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is also presented.

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, ...

Biochemistry

A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often viruses exhibit cell and tissue tropism and are adapted to specific cell or tissue proteases. Moreover, these viruses can introduce mutations or insertions into their genome during replication that may affect the cleavage, and thus can contribute to adaptations to a new host. Here, we present a fluorogenic peptide cleavage assay that allows a rapid screening of peptides mimicking the cleavage site of viral fusion proteins. The technique is very flexible and can be used to investigate the proteolytic activity of a single protease on many different substrates, and in addition, it also allows exploration of the activity of multiple proteases on one or more peptide substrates. In this study, we used peptides mimicking the cleavage site motifs of the coronavirus spike protein. We tested human and camel derived Middle East Respiratory Syndrome coronaviruses (MERS-CoV) to demonstrate that single and double substitutions in the cleavage site can alter the activity of furin and dramatically change cleavage efficiency. We also used this method in combination with bioinformatics to test furin cleavage activity of feline coronavirus spike proteins from different serotypes and strains. This peptide-based method is less labor- and time intensive than conventional methods used for the analysis of proteolytic activity for viruses, and results can be obtained within a single day.

We present a fluorogenic peptide cleavage assay that allows a rapid screening of the proteolytic activity of proteases on peptides representing the cleavage site of viral fusion peptides. This method can also be used on any other amino acid motif within a protein sequence to test for the protease activity.

Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often ...

Use of the Protease Fluorescent Detection Kit to Determine Protease Activity

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect protease activity uses casein labeled with fluorescein isothiocyanate (FITC) as the substrate.
Protease activity results in the cleavage of the FITC-labeled casein substrate into smaller fragments, which do not precipitate under acidic conditions. After incubation of the protease sample and substrate, the reaction is acidified with the addition of trichloroacetic acid (TCA). The mixture is then centrifuged with the undigested substrate forming a pellet and the smaller, acid soluble fragments remaining in solution. The supernatant is neutralized and the fluorescence of the FITC-labeled fragments is measured.
The described kit procedure detects the trypsin protease control at a concentration of approximately 0.5 &mu;g/ml (5 ng of trypsin added to the assay). This sensitivity can be increased with a longer incubation time, up to 24 hours. The assay is performed in microcentrifuge tubes and procedures are provided for fluorescence detection using either cuvettes or multiwell plates.

The Protease Fluorescent Detection Kit is designed for the measurement of protease activity using fluorometry. It is also suitable for detection of trace amounts of protease contamination. The method is based on the proteolytic hydroysis of a proprietary formulation of a FITC-labeled casein substrate.

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect ...

Biology

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 ...

Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established for many membrane proteins. A simple technique called fluorescence protease protection (FPP) is presented, which permits the determination of membrane protein topology in living cells. This technique has numerous advantages over other methods for determining protein topology, in that it does not require the availability of multiple antibodies against various domains of the membrane protein, does not require large amounts of protein, and can be performed on living cells. The FPP method employs the spatially confined actions of proteases on the degradation of green fluorescent protein (GFP) tagged membrane proteins to determine their membrane topology and orientation. This simple approach is applicable to a wide variety of cell types, and can be used to determine membrane protein orientation in various subcellular organelles such as the mitochondria, Golgi, endoplasmic reticulum and components of the endosomal/recycling system. Membrane proteins, tagged on either the N-termini or C-termini with a GFP fusion, are expressed in a cell of interest, which is subject to selective permeabilization using the detergent digitonin. Digitonin has the ability to permeabilize the plasma membrane, while leaving intracellular organelles intact. GFP moieties exposed to the cytosol can be selectively degraded through the application of protease, whereas GFP moieties present in the lumen of organelles are protected from the protease and remain intact. The FPP assay is straightforward, and results can be obtained rapidly.

Determining Membrane Protein Topology Using Fluorescence Protease Protection FPP

Here, we present a protocol to determine the orientation and topology of integral membrane proteins in living cells. This simple protocol relies on selective protease sensitivity of chimeras between the protein of interest and GFP.

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established ...

Zymography Gel Electrophoresis: An Electrophoretic Technique to Detect Matrix Metalloproteinases by Assessing the Enzymatic Activity

Source: Hu, X. et al., <a href="https://www.jove.com/v/2445">Detection of Functional Matrix Metalloproteinases by Zymography</a>. J. Vis. Exp. (2010)
This video demonstrates the zymography gel electrophoresis technique to assess the activity of matrix metalloproteinases (MMPs) present in cell culture supernatant. The active MMPs degrade the MMP-specific substrate embedded in a polyacrylamide gel, which appears as clear bands against a dark stained background following staining.

Source: Hu, X. et al., Detection of Functional Matrix Metalloproteinases by Zymography. J. Vis. Exp. (2010)
This video demonstrates the zymography gel ...

JoVE Encyclopedia of Experiments

Biological Techniques

Electrophoresis Techniques

A Fluorogenic Peptide Cleavage Assay to Screen the Proteolytic Activity of Proteases

Source: Jaimes, J. A., et al. <a href="https://app.jove.com/v/58892">A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation.</a> J. Vis. Exp. (2019).
This video demonstrates an assay to screen for the proteolytic activity of proteases using fluorogenic peptides. The protease recognizes its cleavage site on the peptide, cleaving it and separating the quencher from the fluorophore, enabling its fluorescence emission. The fluorescence signal is detected and analyzed to check for the cleavage efficiency of different peptide variants.

1. Designing and Preparing the Peptides

	Acquire the sequence of the fusion protein of interest from a public database such as NCBI ...

Immunology

Immunodiagnostics

Specialized Assays

Fluorescent Peptide Zymography: A Modified Technique to Detect Protease Activity Using Fluorogenic Substrates in Zymogram Gels

Source: Deshmukh, A. A. et al. <a href="https://www.jove.com/video/58938" target="_blank">Detection of Protease Activity by Fluorescent Peptide Zymography.</a> J. Vis. Exp. (2019)
In this video, we perform zymography, an electrophoretic technique, to analyze the proteolytic activity within biological samples.

Source: Deshmukh, A. A. et al. Detection of Protease Activity by Fluorescent Peptide Zymography. J. Vis. Exp. (2019)
In this video, we perform zymography, ...

Detection of Protease Activity by Fluorescent Peptide Zymography

Samenvatting

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