Zymography Gel Electrophoresis: An Electrophoretic Technique to Detect Matrix Metalloproteinases by Assessing the Enzymatic Activity
Transcripción
Prior to the start of this protocol, prepare all matrix metalloproteinases, or MMPs, adequately to maintain the function of the enzymes. The samples can be used immediately or stored at -80 degrees Celsius. Here MMP-2, also known as gelatinase A or type IV collagenase, is used to demonstrate visualization of MMP activity with zymography.
Once MMP enzymes have been prepared, obtain a 10% gelatin zymogram gel pouch containing a gel inside a cassette. Rinse the cassette with deionized water. Remove the protective tape from the bottom of the cassette, and the comb from the top of the gel. Rinse the wells three times with Tris-Glycine SDS running buffer.
Place the gel into a mini-cell electrophoresis apparatus, ensuring that the smaller side of the cassette faces inward. Lock the gel into place with the Gel Tension Wedge. Fill the top chamber of the apparatus with Tris-Glycine SDS running buffer above the level of the wells, and check for any leaks. Fill the lower chamber of the gel electrophoresis set-up with running buffer as well.
Then, load 10 microliters of protein molecular marker.
Mix an equal amount of the sample to a gel-loading buffer which does not contain reducing agents. Unlike regular SDS-PAGE, do not boil the samples, and instead directly load them into the wells of the gel, using gel-loading tips. These wells can hold up to 20 microliters of volume.
Place the lid on the mini-cell, and connect the electrode cords to the power supply. Switch the power supply on and set the gel to run at 125 volts, constant for 90 minutes. Check the formation of small bubbles on the wire of the lower chamber, indicating current circulation.
When running the first gel, monitor the progress of migration every 15 minutes, using the bromophenol blue included in the loading buffer as an indicator. For each gel, prepare 100 milliliters of 1X zymogram-renaturing buffer, and 200 milliliters of zymogram-developing buffer, both in deionized water.
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 a gel knife. Cut a corner to mark the direction of the gel. Carefully remove the gel from the cassette, and place it into a container with 100 milliliters of renaturing buffer.
Incubate the gel for 30 minutes at room temperature with gentle agitation.
Remove the renaturing buffer, and add 100 milliliters of developing buffer to the gel. Incubate for 30 minutes at room temperature with gentle agitation.
Then, remove the developing buffer, and add an additional 100 milliliters of fresh developing buffer to the gel. Incubate the gel overnight at 37 degrees Celsius.
Following overnight incubation, remove the developing buffer, and wash the gel by incubating it with deionized water, under gentle agitation, for 5 minutes at room temperature. Repeat this wash two times.
Place the gel on a plastic sheet protector, and remove bubbles. Then, scan the gel with a photo-scanner to save the exact positioning of the protein standard bands, as they will become barely visible after gel staining.
Stain the gel by adding 20 milliliters of SimplyBlue SafeStain to the gel. Incubate the gel for 1 hour at room temperature, under gentle agitation.
Remove the SimplyBlue SafeStain, and destain the gel in 100 milliliters of deionized water at room temperature under gentle agitation.
After 1 hour, replace with fresh deionized water, and continue to incubate the gel for at least one hour.
