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

Overview

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.

Protocol

1. Loading and Running the Gel

1. All samples must be prepared adequately to maintain the function of the enzymes and used immediately after collection or stored frozen at -80°C. The samples must not contain reducing agents (such a β-mercaptoethanol) or be boiled before gel loading.
2. Open the pouch containing a gel inside a cassette, rinse the cassette with deionized water.
   1. Remove the protective tape from the bottom of the cassette and the comb from the top of the gel.
   2. Rinse the wells three times with running buffer (diluted to 1X in deionized water).
   3. Place the gel into the Mini-Cell, ensuring that the smaller side of the cassette faces inwards. Lock into place with the Gel Tension Wedge. The Mini-Cell allows to run one or two gels in parallel.
   4. Fill the top (inside) chamber with 1X running buffer above wells level, check for any leaks. In case of leaks, remove the buffer and reposition the gel.
   5. Fill the lower chamber with 1X running buffer.
3. Load 10 μL of protein molecular marker in one well.
   1. Mix an equal amount of gel-loading buffer and of sample and load into the wells of the gel using gel-loading tips (changed between each sample). These wells can be loaded with up to 20 μL total.
4. Place the lid on the Mini-Cell and connect the electrode cords to the power supply. Switch the power supply on and set it to run at 125 V constant for 90 minutes. Check the formation of small bubbles on the wire of the lower chamber, indicating current circulation.
5. When running your first gel, monitor progress of the migration every 15 minutes, using the bromophenol blue included in the loading buffer as an indicator. Let the gel run until the indicator dye reaches the bottom of the gel.

2. Renaturing and Developing the Gel

1. For each gel, prepare 100 mL of 1X renaturing buffer and 200 mL of denaturing buffer, both in deionized water.
2. When the bromophenol blue tracking dye reaches the bottom of the gel, switch the power supply off, open the Mini-Cell, and remove the gel. Separate the two sides of the cassette using the gel knife (or a weighing spatula). Cut a corner to mark the gel's direction.
3. Carefully remove the gel from the cassette and place into a container with 100 mL renaturing buffer. Incubate for 30 minutes at room temperature with gentle agitation.
4. Remove the renaturing buffer and add 100 mL of developing buffer to the gel. Incubate for 30 minutes at room temperature with gentle agitation.
5. Remove the developing buffer and add 100 mL more of developing buffer to the gel. Incubate overnight (16-18 hours) at 37°C.
6. Remove the developing buffer and rinse three times (5 minutes each) with deionized water under gentle agitation at room temperature.
7. Scan the gel to save the exact positioning of the protein standard bands as they will become less or not visible after gel staining.
8. Stain the gel by adding 20 mL of SimplyBlue Safestain to the gel. Incubate for 1 hour at room temperature under gentle agitation.
9. Remove the SimplyBlue SafeStain and de-stain the gel in 100 mL or more deionized water for one hour at room temperature under gentle agitation.
10. For better results, replace with fresh deionized water and incubate for another hour or more at room temperature under gentle agitation.

Materials

Name	Company	Catalog Number	Comments
Xcell SureLock Mini-Cell CE mark electrophoresis apparatus	Invitrogen	EI0001
Power supply (model 302)	VWR	93000-744
Novex 10% gelatin zymogram gels, 1.0 mm, 12 wells	Invitrogen	EC61752BOX
Blue Juice gel loading buffer	Invitrogen	10816015
Gel loading tips	VWR	53509-015
Protein molecular weight standard	Invitrogen	LC5800
Novex Tris-Glycine-SDS running buffer	Invitrogen	LC2675
Novex zymogram renaturing buffer	Invitrogen	LC2670
Novex zymogram developing buffer	Invitrogen	LC2671
SimplyBlue SafeStain	Invitrogen	LC6060