<p class='jove_title'><strong>1. Electrophoresis of Biological Samples in Peptide Zymography Gels</strong></p>
<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&#8211;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).<br />
	<strong>NOTE:</strong> Most MMPs and their variants fall within the range of 35&#8211;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 ZnCl<sub>2</sub>, and 5 mM CaCl<sub>2</sub>&#160;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 ZnCl<sub>2</sub>&#160;and 5 mM CaCl<sub>2</sub>&#160;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>
	<li>After 24 hours, image gels using a fluorescent gel scanner/imager using the appropriate excitation and emission filters.</li>
</ol>

<table><tbody><tr><td>1.5 mm Empty Gel Cassettes&amp;nbsp;</td><td>ThermoFisher Scientific&amp;nbsp;</td><td>NC2015</td><td></td></tr><tr><td>20% SDS Solution&amp;nbsp;</td><td>Ambion&amp;nbsp;</td><td>AM9820</td><td></td></tr><tr><td>3x Zymography Sample Buffer&amp;nbsp;</td><td>Bio-Rad&amp;nbsp;</td><td>1610764</td><td></td></tr><tr><td>Azido-PEG3-Maleimide Kit</td><td>Click Chemistry Tools</td><td>AZ107</td><td></td></tr><tr><td>PowerPac Basic Power Supply&amp;nbsp;</td><td>Bio-Rad&amp;nbsp;</td><td>1645050</td><td></td></tr><tr><td>Precision Plus Protein Dual Color Standard&amp;nbsp;</td><td>Bio-Rad&amp;nbsp;</td><td>161-0374</td><td></td></tr><tr><td>PrecisionGlide Hypodermic Needles&amp;nbsp;</td><td>Fisher Scientific&amp;nbsp;</td><td>14-826</td><td></td></tr></tbody></table>

fluorescent peptide zymography: a modified technique to detect protease activity using fluorogenic substrates in zymogram gels

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

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

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

<p>Zymography is an electrophoretic technique that involves a protein substrate copolymerized with polyacrylamide gel to detect hydrolytic enzymes and their activity. To begin, dissolve samples in a zymography buffer containing glycerol, sodium dodecyl sulfate or SDS, and bromophenol blue.</p>
<p>Glycerol makes the sample buffer denser than the surrounding running buffer of the gel, enabling easy loading into the gel pockets. SDS - an anionic detergent - denatures enzymes in the sample and coats them with a negative charge. Bromophenol blue - a tracking dye - helps monitor the sample progress in the gel.</p>
<p>Load the samples in polyacrylamide gel embedded with fluorogenic substrates to detect protease activity. Now, connect the electrodes and begin electrophoresis. The generated electric field causes all the SDS-bound enzymes to migrate through the gel towards the positively charged electrode.</p>
<p>Following completion of electrophoresis, remove the gel and wash it in a renaturing buffer containing non-ionic detergent that displaces SDS to allow enzyme renaturation. Next, incubate the gel in a developing buffer containing divalent cations that activate the enzymes in the gel.</p>
<p>The active proteases cleave the fluorogenic substrates, causing an increase in the fluorescence. Image the gel to observe fluorescent protein bands whose intensity is proportional to protease activity.</p>

<p>To begin, dissolve samples in conventional zymography sample buffer. Add 400 milliliters of 1x Tris-Glycine SDS running buffer to the gel apparatus. Load up to 35 microliters of sample per well. Run the samples at 120 volts at 4 degrees Celsius for 1.5 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.</p>
<p>Wash the gels three times in renaturing 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.</p>

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2.9K Views. Zymography is an electrophoretic technique that involves a protein substrate copolymerized with polyacrylamide gel to detect hydrolytic enzymes and their activity. To begin, dissolve samples in a zymography buffer containing glycerol, sodium dodecyl sulfate or SDS, and bromophenol blue. Glycerol makes the sample buffer denser than the surrounding running buffer of the gel, enabling easy loading into the gel pockets. SDS - an anionic detergent - denatures enzymes in the sample and coats...

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<table><tbody><tr><td>1.5 mm Empty Gel Cassettes&nbsp;</td><td>ThermoFisher Scientific&nbsp;</td><td>NC2015</td><td></td></tr><tr><td>20% SDS Solution&nbsp;</td><td>Ambion&nbsp;</td><td>AM9820</td><td></td></tr><tr><td>3x Zymography Sample Buffer&nbsp;</td><td>Bio-Rad&nbsp;</td><td>1610764</td><td></td></tr><tr><td>Azido-PEG3-Maleimide Kit</td><td>Click Chemistry Tools</td><td>AZ107</td><td></td></tr><tr><td>PowerPac Basic Power Supply&nbsp;</td><td>Bio-Rad&nbsp;</td><td>1645050</td><td></td></tr><tr><td>Precision Plus Protein Dual Color Standard&nbsp;</td><td>Bio-Rad&nbsp;</td><td>161-0374</td><td></td></tr><tr><td>PrecisionGlide Hypodermic Needles&nbsp;</td><td>Fisher Scientific&nbsp;</td><td>14-826</td><td></td></tr></tbody></table>

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

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