Name: One-step Extraction and Zymographic Analysis of Bacterial Gelatinases (Video) | JoVE Journal
Uploaded: 2025-08-01T12:40:00.000+00:00
Duration: 7 min 20 s
Description: Learn a one-step extraction method for active gelatinases from Leptospira using zymography and LC-MS/MS. This streamlined protocol enables pathogenesis research and biomarker discovery for leptospirosis detection.

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5 minutes

The authors express their gratitude to SERB Division, Department of Science and Technology, New Delhi, for their support through grant No.SR/S0/HS/0108/2012, to MGM

1. Extraction of bacterial gelatinases
NOTE: See Table 1 for preparation of reagents and materials.
<ol>
	<li>Grow the organism in Ellinghausen McCullough Johnson Harris (EMJH) medium containing 10 mg/mL bovine serum albumin at 30 &#176;C. Take 10 mL of Leptospira culture in the mid-log phase of growth containing 3.5 &#215; 108 cells/mL for the extraction proteinases13,14. Use the direct microscopic count with a Petroff-Hausser counting chamber under a dark-field microscope to observe the mid-log phase of growth.</li>
	<li>Harvest the cells by centrifugation at 6,000 &#215; g for 10 min, and discard the supernatant in a container for decontamination by autoclaving at 121 &#176;C for 60 min.</li>
	<li>Transfer the pellet by suspending it in 1 mL of wash buffer to a 1.5 mL microcentrifuge tube and centrifuge at 6,000 &#215; g for 5 min. Repeat this wash.</li>
	<li>Extract the protein from the pellet by adding 100 &#181;L of extraction buffer for a Leptospira pellet from 10 mL of culture and vortex for 5 min. Continue the extraction by incubating at 4 &#176;C for 1 h. Collect the extracted protein by centrifugation at 15,000 &#215; g at 4 &#176;C for 10 min, and discard the pellet in a container for decontamination by autoclaving at 121 &#176;C for 60 min.</li>
	<li>Use the protein extract directly for zymography by adding an equal volume of 2x sample buffer.</li>
	<li>Use the Bradford estimation assay to determine the protein concentration.</li>
	<li>If the protein concentration is low, precipitate it with acetone by mixing the protein with a 4x volume of acetone for precipitation and incubate it overnight at -20 &#176;C. The next day, centrifuge the sample at 10,000 &#215; g for 10 min, air dry the resulting pellet, and resuspend it in PBS or dissolve in the desired buffer and store it at -20 &#176;C for up to 1 month. Use lyophilization for long-term storage or transport. 
	NOTE: Due to active proteases in the extract and the absence of protease inhibitors, proteins may degrade in the sample.</li>
</ol>
2. Zymography of gelatinases
<ol>
	<li>Preparation of the gelatin co-polymerized SDS PAGE gel
	<ol>
		<li>Resolving gel
		<ol>
			<li>Make the resolving gel mix by adding the components in the required amounts to cast the gel. To 3.86 mL of water in a 50 mL beaker, add 2.5 mL of Tris-HCl (pH 8.8), 0.1 mL from the 10% gelatin stock solution, 3.34 mL of acrylamide-bis acrylamide solution (ABA), 0.1 mL of 10% SDS, 0.006 mL of TEMED, and 0.1 mL of 10% ammonium persulfate and mix well without foaming and bubbles. The system mentioned (Regular Glass Plates; 18 x 8 cm) requires 10 mL of resolving gel mix. 
			NOTE: If the gelatin is from a frozen aliquot, thaw it till it melts to clear the solution without any clumps. Add ammonium persulfate just before casting because the polymerization will start immediately after adding ammonium persulfate.</li>
			<li>Pour the resolving gel mix into the casting chamber up to ~3-5 mm below the marking for the bottom of the wells. Avoid air bubbles.</li>
			<li>Slowly pour some water to obtain an even 1 mm layer on the top of the gel and remove bubbles. Allow the gel to be set for 1 h. 
			NOTE: Gel formation can be confirmed by looking for a phase separation between the layer of water and the gel; the gel cannot be poured out even if the gel cassette is tilted.</li>
		</ol>
		</li>
		<li>Stacking gel
		<ol>
			<li>Prepare the stacking gel by mixing the following components. To 1.84 mL of water in a 10 mL beaker, add 2.5 mL of Tris-Cl (pH 6.6), 0.35 mL of ABA, 0.03 mL of 10% SDS, and 0.006 mL of TEMED. The 8 x 10 cm system of 1 mm thickness requires 3 mL of resolving gel mix.</li>
			<li>From the gel cassette, decant the water formed at the top of the gel by tilting the gel setup to one side and blotting the collected water from the corner using a narrow strip of filter paper.</li>
			<li>Add 0.03 mL of 10% APS to the mixture mentioned in the step 2.1.2.1 and mix without forming bubbles.</li>
			<li>Pour the stacking gel onto the resolving gel and immediately place the comb in position without forming air bubbles under the comb teeth, leaving a thickness of 3-5 mm of stacking gel between the resolving gel and the bottom of the comb teeth. Wait for at least 3 h for complete polymerization and crosslinking of the gelatin.</li>
			<li>Take out the lower spacer and fix the gel on the electrophoresis apparatus.</li>
			<li>Slowly place the gel in the tank containing 1x electrode buffer, ensuring there is no trapped air in the bottom of the gel.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Preparation of sample
	<ol>
		<li>Add an equal volume of sample buffer to the extract and incubate at 37 &#176;C for 30 min to allow SDS to bind to the protein. Use a sample containing 2 &#181;L of human serum with an equal volume of sample buffer as a positive control.</li>
	</ol>
	</li>
	<li>Loading the sample
	<ol>
		<li>Carefully load the sample into the wells with a narrow-tipped pipette or a Hamilton syringe (0.5-1 mL). Gradually add electrode buffer to fill the remaining portions of the wells, the top of the gel, and the upper tank of the electrophoresis unit without disturbing the loaded samples. 
		NOTE: The Hamilton syringe should be thoroughly washed between the samples to avoid cross-contamination.</li>
	</ol>
	</li>
	<li>Running electrophoresis
	<ol>
		<li>Start the electrophoresis at 6 mA per gel and run until the dye front enters the resolving gel. Then, increase the current to 12 mA per gel and continue until the dye front reaches 1 cm above the bottom of the gel. 
		NOTE: The running time may change depending on the concentration of ABA used and the size of the target protein.</li>
	</ol>
	</li>
	<li>Marking the orientation of the bands of the standard
	<ol>
		<li>After completing the electrophoresis, carefully dismantle the glass plates using a plate separation tool or plastic spatula and mark the orientation of sample loading by cutting the right lower corner of the gel and mark the prestained marker bands by punching holes nearby or scanning the gel.</li>
	</ol>
	</li>
	<li>Renaturation of resolved enzymes
	<ol>
		<li>Submerge the gel in renaturing solution containing Triton X-100 in a gel tray and incubate for 30 min at room temperature without shaking. 
		NOTE: Triton X-100 renatures the proteins, as the zymogram gel containing resolved proteins may be denatured and lose activity in the presence of SDS.</li>
	</ol>
	</li>
	<li>Incubation for protease activity
	<ol>
		<li>Replace the renaturing solution with an activation buffer that completely submerges the gel and incubate at 37 &#176;C for 18 h in an incubator. 
		NOTE: Modify the activation buffer by adding inhibitors or activators of proteinases of interest to characterize proteinase activity as needed.</li>
	</ol>
	</li>
	<li>Staining of the zymogram
	<ol>
		<li>After incubation, remove the activation buffer and rinse the gel briefly with water for 1 min to remove any remaining Triton X-100 and to prevent foaming during shaking.</li>
		<li>Add the staining solution to the tray, ensuring the gel is completely submerged. Shake the gel gently at a low speed to prevent damage. Continue staining until the gel turns completely blue.</li>
		<li>Remove excess stain from the gel surface by rinsing it with water for 1 min. Add the destaining solution to the tray and shake gently. Replace the destaining solution with fresh solution several times until the desired band contrast is achieved against the blue background. 
		NOTE: While performing the destaining, continuously monitor the gel to ensure it does not become completely destained, allowing observation and recording of the proteolytic bands.</li>
		<li>Scan the gel in a gel documentation system or document scanner, and determine the molecular mass of the protein bands by comparing them to the protein standards.</li>
	</ol>
	</li>
</ol>

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<li>Ambili, M., Jayasree, K., Sudhakaran, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((60K+gelatinase+involved+in+mammary+gland+involution+is+regulated+by+%CE%B2-oestradiol%5BTitle%5D)+AND+%22Biochim+Biophys+Acta%22%5BJournal%5D)">60K gelatinase involved in mammary gland involution is regulated by β-oestradiol.</a> Biochim Biophys Acta. 1403 (3), 219-231 (1998).</li>
<li>Zhang, Y. -Z., Ran, L. -Y., Li, C. -Y., Chen, X. -L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Diversity%2C+structures%2C+and+collagen-degrading+mechanisms+of+bacterial+collagenolytic+proteases%5BTitle%5D)+AND+%22Appl+Environ+Microbiol%22%5BJournal%5D)">Diversity, structures, and collagen-degrading mechanisms of bacterial collagenolytic proteases.</a> Appl Environ Microbiol. 81 (18), 6098-6107 (2015).</li>
<li>Duarte, A. S., Correia, A., Esteves, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Bacterial+collagenases+-+A+review%5BTitle%5D)+AND+%22Crit+Rev+Microbiol%22%5BJournal%5D)">Bacterial collagenases - A review.</a> Crit Rev Microbiol. 42 (1), 106-126 (2016).</li>
<li>Madathiparambil, M. G., Cattavarayane, S., Perumana, S. R., Manickam, G. D., Sehgal, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Presence+of+46+kDa+gelatinase+on+the+outer+membrane+of+Leptospira%5BTitle%5D)+AND+%22Curr+Microbiol%22%5BJournal%5D)">Presence of 46 kDa gelatinase on the outer membrane of Leptospira.</a> Curr Microbiol. 62 (5), 1478-1482 (2011).</li>
<li>Kramer, R. A., Zandwijken, D., Egmond, M. R., Dekker, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vitro+folding%2C+purification+and+characterization+of+Escherichia+coli+outer+membrane+protease+OmpT%5BTitle%5D)+AND+%22Eur+J+Biochem%22%5BJournal%5D)">In vitro folding, purification and characterization of Escherichia coli outer membrane protease OmpT.</a> Eur J Biochem. 267 (3), 885-893 (2000).</li>
<li>Kum, S. L., Ho, J. C. S., Parikh, A. N., Liedberg, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Amphiphilic+membrane+environments+regulate+enzymatic+behaviors+of+Salmonella+outer+membrane+protease%5BTitle%5D)+AND+%22ACS+Bio+Med+Chem+Au%22%5BJournal%5D)">Amphiphilic membrane environments regulate enzymatic behaviors of Salmonella outer membrane protease.</a> ACS Bio Med Chem Au. 2 (1), 73-83 (2022).</li>
<li>Sarma, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Extracellular+proteome+analysis+shows+the+abundance+of+histidine+kinase+sensor+protein%2C+DNA+helicase%2C+putative+lipoprotein+containing+peptidase+M75+domain+and+peptidase+C39+domain+protein+in+Leptospira+interrogans+grown+in+EMJH+medium%5BTitle%5D)+AND+%22Pathogens%22%5BJournal%5D)">Extracellular proteome analysis shows the abundance of histidine kinase sensor protein, DNA helicase, putative lipoprotein containing peptidase M75 domain and peptidase C39 domain protein in Leptospira interrogans grown in EMJH medium.</a> Pathogens. 10 (7), 852(2021).</li>
<li>Seddon, A. M., Curnow, P., Booth, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Membrane+proteins%2C+lipids+and+detergents%3A+not+just+a+soap+opera%5BTitle%5D)+AND+%22Biochim+Biophys+Acta%22%5BJournal%5D)">Membrane proteins, lipids and detergents: not just a soap opera.</a> Biochim Biophys Acta. 1666 (1-2), 105-117 (2004).</li>
<li>Levy, D., Gulik, A., Seigneuret, M., Rigaud, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Phospholipid+vesicle+solubilization+and+reconstitution+by+detergents.+Symmetrical+analysis+of+the+two+processes+using+octaethylene+glycol+mono-n-dodecyl+ether%5BTitle%5D)+AND+%22Biochemistry%22%5BJournal%5D)">Phospholipid vesicle solubilization and reconstitution by detergents. Symmetrical analysis of the two processes using octaethylene glycol mono-n-dodecyl ether.</a> Biochemistry. 29 (40), 9480-9488 (1990).</li>
<li>Lichtenberg, D., Ahyayauch, H., Alonso, A., Goñi, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Detergent+solubilization+of+lipid+bilayers%3A+a+balance+of+driving+forces%5BTitle%5D)+AND+%22Trends+Biochem+Sci%22%5BJournal%5D)">Detergent solubilization of lipid bilayers: a balance of driving forces.</a> Trends Biochem Sci. 38 (2), 85-93 (2013).</li>
<li>Schägger, H., Cramer, W. A., von Jagow, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Analysis+of+molecular+masses+and+oligomeric+states+of+protein+complexes+by+blue+native+electrophoresis+and+isolation+of+membrane+protein+complexes+by+two-dimensional+native+electrophoresis%5BTitle%5D)+AND+%22Anal+Biochem%22%5BJournal%5D)">Analysis of molecular masses and oligomeric states of protein complexes by blue native electrophoresis and isolation of membrane protein complexes by two-dimensional native electrophoresis.</a> Anal Biochem. 217 (2), 220-230 (1994).</li>
<li>Schägger, H., von Jagow, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Blue+native+electrophoresis+for+isolation+of+membrane+protein+complexes+in+enzymatically+active+form%5BTitle%5D)+AND+%22Anal+Biochem%22%5BJournal%5D)">Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form.</a> Anal Biochem. 199 (2), 223-231 (1991).</li>
<li>Zeug, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Current+microscopic+methods+for+the+neural+ECM+analysis%5BTitle%5D)+AND+%22Prog+Brain+Res%22%5BJournal%5D)">Current microscopic methods for the neural ECM analysis.</a> Prog Brain Res. 214, 287-312 (2014).</li>
<li>Madathiparambil, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+zymography+analysis+of+proteinase+activity+present+in+Leptospira%5BTitle%5D)+AND+%22Curr+Microbiol%22%5BJournal%5D)">A zymography analysis of proteinase activity present in Leptospira.</a> Curr Microbiol. 62 (3), 917-922 (2011).</li>
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The authors have no conflicts of interest to declare.

During the preparation of the acrylamide gel, gelatin co-polymerizes with the acrylamide polymer matrix. Consequently, the co-polymerized gelatin remains immobilized within the gel and does not migrate during electrophoresis, even though it is a protein. After electrophoresis, the native proteins extracted using deoxycholate and 6-aminocaproic acid buffer separate according to their mass and charge. After removing the SDS, these proteins become renatured in the presence of Triton X-100 and regain their activity. Under na...

Most bacterial pathogens must encounter the host&#39;s extracellular matrix (ECM) during invasion and dissemination to target organs during pathogenesis. Collagen forms the major component in the ECM of animal tissues1. Gelatinases are a group of proteases that act on collagen and its hydrolyzed form, gelatin. The ECM degradation is primarily executed by a group of proteases, which includes collagenases and gelatinases2,3. Bacterial pathogens use collagenases and gelatinases to remodel or break down the host ECM for their convenience in invasion and pathogenesis4,5. Bacterial ECM-degrading enzymes are potential candidates for vaccine and drug targeting5.
Bacterial proteases are found in secretory and membrane-bound forms6,7,8,9. Many methods for cellular enzyme extraction are ineffective for isolating membrane-bound bacterial gelatinases and collagenases, as these procedures may not efficiently preserve the native form of membrane-bound proteins. Similarly, there are membrane-specific methods that extract only membrane-bound proteases and gelatinases. Conventional extraction methods for total proteins, including membrane proteins, often involve detergents and chaotropic ions, which can impact enzymatic activity. An extraction procedure that maintains enzyme activity and solubility is essential for electrophoretic separation and enables zymography development to detect protease activity.
Sodium deoxycholate, an ionic detergent that can lyse cells by disrupting the cell membrane, enables the release of the cell contents and membrane protein. The 6-aminocaproic acid can solubilize the released membrane protein in its native form. Solubilization by 6-aminocaproic acid has an advantage that it does not affect electrophoresis. Here, we demonstrate the extraction of cellular and membrane-bound bacterial proteinases using sodium deoxycholate and 6-aminocaproic acid, stabilizing them in their native soluble form10,11,12. This method effectively solubilizes proteins from the cell and the membrane that can be used directly without interfering electrophoresis13,14. Various enzymatic characterizations can be performed by changing the protease inhibitors and metal ions used in the activation buffer, depending on the application.

<table><tbody><tr><td>6-Aminocaproicacid</td><td>Merck</td><td>A2504</td><td></td></tr><tr><td>Acetic acid</td><td>&amp;nbsp; Sigma</td><td>&amp;nbsp;695092</td><td></td></tr><tr><td>Acetone</td><td>&amp;nbsp;&amp;nbsp;Sigma</td><td>&amp;nbsp;32201</td><td></td></tr><tr><td>Acrylamide</td><td>&amp;nbsp;&amp;nbsp;Sigma</td><td>&amp;nbsp;A-3553</td><td></td></tr><tr><td>Ammonium persulfate</td><td>&amp;nbsp;&amp;nbsp;Sigma</td><td>&amp;nbsp;A-3678</td><td></td></tr><tr><td>Bis Acrylamide</td><td>&amp;nbsp;&amp;nbsp;Sigma</td><td>&amp;nbsp;M7279</td><td></td></tr><tr><td>Bovine serum albumin fraction V (BSA)</td><td>&amp;nbsp; Sigma</td><td>&amp;nbsp;A-8022</td><td></td></tr><tr><td>Bromophenol Blue</td><td>&amp;nbsp;&amp;nbsp;Sigma</td><td>&amp;nbsp;B-8026</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Merck</td><td>C4901</td><td></td></tr><tr><td>Coomassie brilliant blue</td><td>&amp;nbsp;&amp;nbsp;Sigma</td><td>1.12553</td><td></td></tr><tr><td>Deoxycholate</td><td>&amp;nbsp;&amp;nbsp;Sigma-Aldrich</td><td>D6750</td><td></td></tr><tr><td>Ellinghausen&amp;ndash;McCullough&amp;ndash;Johnson&amp;ndash;Harris (EMJH) medium, Difco</td><td>&amp;nbsp;Difco</td><td>&amp;nbsp;279410</td><td></td></tr><tr><td>Gelatin</td><td>Merck</td><td>G9382</td><td></td></tr><tr><td>Glycerol</td><td>Merck</td><td>G5516</td><td></td></tr><tr><td>Glycine G8790</td><td>&amp;nbsp;&amp;nbsp;Sigma</td><td>&amp;nbsp;G-8898</td><td></td></tr><tr><td>Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 (ATCC BAA-1198)&amp;nbsp;, maintained in the biorepository ofICMR-RMRC Portblair</td><td>Maintained in the biorepository of ICMR-RMRC Port Blair</td><td></td><td>Optimum growth temperature 30 &amp;deg;C</td></tr><tr><td>Magnesium chloride M8266</td><td>&amp;nbsp; Sigma</td><td>&amp;nbsp;M8266</td><td></td></tr><tr><td>Methanol</td><td>&amp;nbsp;&amp;nbsp;Sigma</td><td>&amp;nbsp;17995-7</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>&amp;nbsp;&amp;nbsp;Sigma</td><td>P4417&amp;nbsp;</td><td></td></tr><tr><td>Plate separation tool</td><td>Wonder Wedgle</td><td></td><td></td></tr><tr><td>Rotor</td><td>Thermoscientific</td><td>FIBERLite F15 8x50 cy</td><td></td></tr><tr><td>Sodium dodecyl sulfate (SDS)</td><td>&amp;nbsp;&amp;nbsp;Sigma</td><td>&amp;nbsp;L-4396</td><td></td></tr><tr><td>TEMED</td><td>&amp;nbsp;&amp;nbsp;Sigma</td><td>&amp;nbsp;T-9281</td><td></td></tr><tr><td>Triton X-100</td><td>&amp;nbsp;&amp;nbsp;Sigma-Aldrich</td><td>T8787</td><td></td></tr><tr><td>Trizma Base (TRIS)&amp;nbsp;</td><td>&amp;nbsp;&amp;nbsp;Sigma</td><td>&amp;nbsp;T6066</td><td></td></tr></tbody></table>

one-step extraction and zymographic analysis of bacterial gelatinases

One-step Extraction and Zymographic Analysis of Bacterial Gelatinases

Proteinases of pathogenic bacteria contribute to invasion and pathogenesis by actively degrading the host's extracellular matrix (ECM) components and cellular barriers, modifying the host immune response and degrading the host defense system. This makes the proteinases one of the most virulent molecules and their identification and characterization are essential for preventing and controlling disease. Many bacteria also possess outer membrane-bound proteinases. Isolation and characterization of outer membrane proteins in their active forms present significant challenges due to their localization within the outer membrane. In this study, we demonstrate a single-step method for extracting both cytoplasmic and outer membrane-associated gelatinases from Leptospira and demonstrate their enzymatic activity using zymography. In this method, we use sodium deoxycholate to disrupt the cell membrane and release the bound proteins and 6-aminocaproic acid to solubilize the released membrane protein in its native form. Further, we show a zymographic analysis of the leptospiral gelatinases. This approach provides a versatile framework for isolating, purifying, and characterizing metalloproteases, collagenases, and gelatinases from various bacterial pathogens.

The result shows the zymogram of leptospiral protein and gelatinases from human serum. The results, shown in Figure 1, represent proteases as white clear bands against a blue background. The bands represent areas where the copolymerized gelatin is degraded and hence, not stained, while other areas of the entire gel are stained blue due to the presence of gelatin. Further, it shows that even though gelatin is also a protein, it does not get eluted in the electrophoresis due to copolymerizatio...

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.

Proteinases of pathogenic bacteria contribute to invasion and pathogenesis by actively degrading the host's extracellular matrix (ECM) components and ...

This research investigates a simpler methodology for extracting and characterizing active gelatinases from Leptospira, utilizing zymography to confirm their enzymatic activity, and addressing challenges in isolating membrane proteins from bacteria. Current technologies include mass spectrometry-based proteomics, two-dimensional gel electrophoresis, bioinformatic tools, zymography, and LC-MS/MS, enabling detailed protein profiling, functional analysis, and identification of virulence factors in Leptospira, and vaccine candidate discovery. Characterizing active outer membrane proteins such as Leptospira gelatinases poses a challenge, particularly due to the membrane localization, which requires efficient disruption and solubilization methods to maintain the protein's integrity and accuracy.This protocol addresses the problem of extracting active outer membrane gelatinases from Leptospira by providing a one-stop extraction process for use in pathogenesis investigation. Our laboratory focuses on leveraging advanced high-resolution LC-MS/MS to systematically identify outer membrane and secretory proteins, particularly focusing on proteinases involved in the extracellular matrix degradation. By elucidating their roles in pathogenesis, we aim to identify sensitive and specific biomarkers for the early-stage detection of leptospirosis.To begin, grow the organism in Ellinghausen-McCullough-Johnson-Harris medium containing 10 milligrams per milliliter of BSA at 30 degrees Celsius. Take 10 milliliters of Leptospira culture in the mid-logarithmic phase of growth, containing 3.5 times 10 to the power of 8 cells per milliliter for proteinase extraction. Centrifuge the culture at 6, 000G for 10 minutes.Discard the supernatant into a container for decontamination. Suspend the pellet in one milliliter of wash buffer, and transfer it to a 1.5 milliliter microcentrifuge tube. Centrifuge the tube at 6, 000G for five minutes, and repeat the wash once more.Now, add 100 microliters of extraction buffer to the Leptospira pellet, and vortex for five minutes. Incubate the mixture at four degrees Celsius for one hour. Then, centrifuge the suspension at 15, 000G at four degrees Celsius for 10 minutes.Post-centrifugation, collect the extracted protein. Prepare the resolving gel mix for sodium dodecyl sulfate-polyacrylamide gel electrophoresis with copolymerized gelatin. Pour the resolving gel mix into the casting chamber up to approximately three to five millimeters below the mark for the bottom of the wells.Slowly pour water to form an even one millimeter layer over the gel, and remove any bubbles, and let the gel set for one hour. Before preparing the stacking gel, tilt the setup to decant the water from the top of the set resolving gel, and blot with a narrow strip of filter paper. Add 0.03 milliliters of 10%ammonium persulfate to the stacking gel mix, and stir gently to avoid bubble formation.Now, pour the stacking gel mix onto the resolving gel and immediately place the comb without trapping air bubbles. Next, fix the gel into the electrophoresis apparatus and remove the lower spacer. After placing the gel in the tank, add an equal volume of sample buffer to the protein extract.Prepare a positive control with two microliters of human serum with an equal volume of sample buffer. Incubate the samples at 37 degrees Celsius for 30 minutes to allow SDS to bind to the protein. Now, carefully load the prepared samples and control into the gel wells using a narrow-tipped pipette.Start the electrophoresis at six milliamperes per gel until the dye front enters the resolving gel. Then increase the current to 12 milliamperes per gel, and continue running until the dye front reaches one centimeter above the bottom of the gel. After electrophoresis, carefully dismantle the glass plates using a plate separation tool or plastic spatula.Mark the orientation of sample loading by cutting the right lower corner of the gel, and then mark the pre-stained marker bands. Submerge the gel in a renaturation solution containing Triton X-100 in a gel tray, and incubate at room temperature for 30 minutes without shaking. Replace the renaturation solution with activation buffer, and incubate again for 18 hours.After removing the activation buffer, rinse the gel with water for one minute, and add the staining solution, ensuring the gel is fully submerged. Gently shake the gel at low speed until the gel turns completely blue. Now, rinse the gel surface with water for one minute to remove excess stain, and add the de-staining solution.Replace the solution several times until the desired contrast is achieved. Finally, scan the gel using a gel documentation system or document scanner to compare the protein bands with the standard markers. The zymogram of Leptospira protein and gelatinases from human serum shows proteases as white clear bands against a blue background.The bands represent areas where the copolymerized gelatin is degraded, and hence, not stained, while other areas of the entire gel are stained blue due to the presence of gelatin.

one-step-extraction-and-zymographic-analysis-of-bacterial-gelatinases

Research

JoVE Journal

Biochemistry

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

Detection of Functional Matrix Metalloproteinases by Zymography

A straight-forward set of methods to isolate and determine the identity of the most abundant proteins expressed in skeletal muscle.  About 800 spots are discerned on a two-dimensional gel from 10 mg muscle; this allows for the determination of gender-specific protein expression.  These methods will give equivalent results in most tissues.

Skeletal Muscle Gender Dimorphism from Proteomics

We have recently reported a novel approach for generating fluorogenic DNAzyme probes that can be applied to set up a simple, "mix-and-read" fluorescent assay for bacterial detection. These special DNA probes catalyze the cleavage of a chromophore-modified DNA-RNA chimeric substrate in the presence of crude extracellular mixture (CEM) produced by a specific bacterium, thereby translating bacterial detection into fluorescence signal generation. In this report we will describe key experimental...

Detection of Bacteria Using Fluorogenic DNAzymes

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation.  Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts 

In this video, we perform zymography, an electrophoretic technique, to analyze the proteolytic activity within biological samples.

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

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.

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

Members of the Burkholderia genus are pathogens of clinical importance.  We describe a method for total bacterial protein extraction, using mechanical disruption, and 2-D gel electrophoresis for subsequent proteomic analysis.

Total Protein Extraction and 2-D Gel Electrophoresis Methods for Burkholderia Species

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.

Detection of Protease Activity by Fluorescent Peptide Zymography

Trehalose analogues are emerging as important molecules for bio(techno)logical and biomedical applications. We describe an optimized protocol for enzymatically synthesizing and purifying trehalose analogues that is simple, efficient, fast, and environmentally friendly. Its application to the rapid production and administration of a probe for the detection of mycobacteria is demonstrated.

Rapid One-step Enzymatic Synthesis and All-aqueous Purification of Trehalose Analogues

The protein composition of the human mitral valve is still partially unknown, because its analysis is complicated by low cellularity and therefore by low protein biosynthesis. This work provides a protocol to efficiently extract protein for the analysis of the mitral valve proteome.

Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve

Learn a one-step extraction method for active gelatinases from Leptospira using zymography and LC-MS/MS. This streamlined protocol enables pathogenesis research and biomarker discovery for leptospirosis detection.

One-step Extraction and Zymographic Analysis of Bacterial Gelatinases

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