The overall goal of this procedure is to detect functional matrix metalloprotein ais. This is achieved by first preparing the sample for zy by adding a loading buffer free of reducing agents agility. Gelatin, ZY Graham gel is then loaded with the sample and allowed to run until the tracking dye reaches the end of the gel.
Following electrophoresis, the gel is re natured, developed, stained and de stain. The final step of the procedure is to scan the gel for data analysis. Ultimately, results can be obtained that show the activity of matrix metalloproteinase in the samples through band density analysis.
Hi, I'm from the laboratory of Dr.Kristin bitten in the Department of Molecular Physiology and biophysics at the LER College of Medicine. Today we'll show you a procedure for detecting matrix metallo Persis by phy. We use this procedure in our laboratory to study matrix Metallo Persis secreted by cultured primary cell.
So let's get studied. Prior to the start of this protocol, prepare all matrix metalloproteinase or MMPs adequately to maintain the function of the enzymes. The samples can be used immediately or stored at minus 80 degrees Celsius.
Here MMP two, also known as gelatinize A or type four collagenase, is used to demonstrate visualization of MP activity with synography. Once MMP enzymes have been prepared, obtain a 10%gelatin zy gram 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 in the first gel. Monitor the progress of migration every 15 minutes using the bromo phenol blue included in the loading buffer as an indicator for each gel.
Prepare 100 milliliters of one time xmo gram Rena buffer, and 200 milliliters of xmo gram developing buffer, both in deionized water. When the bromo phenol 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 Rena Buffer.
Incubate the gel for 30 minutes At room temperature with gentle vegetation, remove the Rena buffer and add 100 milliliters of developing buffer to the gel. Incubate for 30 minutes at room temperature with gentle vegetation. 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 and a gentle vegetation. For five 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 simply blue safe stain to the gel. Incubate the gel for one hour at room temperature. Under gentle agitation, remove the simply blue safe stain and detain the gel in 100 milliliters of deionized water at room temperature under gentle agitation.
After one hour, replace with fresh deionized water and continue to incubate the gel for at least one hour. To analyze the data using cytometry first carefully remove the gel from the water and place it in a plastic sheet protector. Using a photo scanner, scan the gel with a resolution of 300 dots per inch or higher.
Save the image in a TIFF format. Open the TIFF file in Image J or similar quantification software to measure band intensities. Visualize the image in black and white by selecting image type eight bit.
Use the rectangular selection tool to outline the first band drawing a rectangle that is at least twice higher than it is wide. Select analyze gels. Select first lane, and the band will be selected.
Then a new rectangle will appear. Move it to the next band and select Analyze gels, select next lane. Repeat this process until all the bands are selected.
Select analyze gels plot lanes to generate the profile plot for each band. Use the straight line selection to draw baselines so that the peak of interest is a completely enclosed area. Select the wand tool and click inside each peak to select it.
Select analyze gels label peaks to obtain a table with the area for each selected peak. These data can be plotted as such or can be normalized to the value of a chosen band. This normalization is especially important when pooling values from several replicate gels to determine statistical significance of the results.
Each well of the gel shown here was loaded with a different cell culture. Supinate containing different amounts of MMP two. Direct observation of this gel shows obvious differences in MMP two concentrations between some of the wells.
For example, it is clear that both wells four and five contain much more MMP two than wells 10 and 11 for objective quantification of bands. Dense cytometry was used with the Image J software. This software revealed an approximate fourfold difference in mmp.
Two amounts between, well, 11 and wells four and five. Similarly, a twofold difference in MMP two amounts was observed between wells 10 and wells four and five. We are just showing you how to detect functional metrics.
Metal persis by xog. When you do this procedure, it is important to remember not to boil your sample. Otherwise you cannot see any bands on your gel.
So that's it. Thanks for watching and good luck with your experiment.
