My name is David Oppenheimer and I work in the Department of Biology at the University of Florida. Purchasing pre poured poly acrylamide gels for routine protein electrophoresis is convenient because you can always have gels on hand that are ready for use. However, commercial gels can be expensive if your lab runs a lot of gels.
Also, commercial gels are typically poured in plastic cassettes that are designed to be discarded after use, thus generating plastic waste that usually ends up in a landfill. Today we will show you how to reuse these plastic cassettes to pour your own poly acrylamide gels that can be stored for weeks, thus providing your lab with a source of inexpensive ready-made gels for routine protein electrophoresis. Okay, let's get started.
Prepare clean in Vitrogen new page, Novak mini gel cassettes. Remove about one teaspoon of epoxy and completely mix the solution with a Pipette tip. When you have Finished mixing the epoxy, apply the epoxy on the back of the front cassette where the front and back will come in contact.
When you finish applying the epoxy, Make sure there are no breaks in the line. Insert a sheet of paper about the thickness of the bottom opening and place the back of the cassette on top. Move the paper and this prevents any epoxy from entering the bottom opening binder.
Clip the edges for a tight seal and let the cassette sit overnight for a full cure. To Prepare the resolving gel, add 2.25 mils of Forex triase resolving gel buffer at pH of 8.7 in a side arm flask. Then add 3.50 Mils of 30.8%acrylamide.
Then add 3.25 mils of double distilled water for a total volume of nine mils. Dega the solution for 10 minutes. When The solution is done to gassing, remove nine mils of the acrylamide solution to a 50 mil orange cap tube.
Then Add 30 microliters of 10%a PS with six microliters of temid. To start the polymerizing process, Make sure to mix well Before you start pouring the resolving gel. Make sure you cover the bottom opening with electrical tape.
Then pour the solution inside the cassette, allowing it to run down the side to prevent any bubbles from forming. Stop pouring when you're about one centimeter from the top. Then pour 0.5 mils of one butanol saturated with one x resolving gel buffer to completely cover the top.
Prepare the stacking gel the same way you prepare the resolving gel. In a sidearm flask, add Forex tris based stacking gel buffer with 30.8%acrylamide and double the distilled water for a total volume of three mils. Dega for 10 minutes and add nine microliters of 10%a PS and two microliters of temid.
And make sure to mix well After one hour. The one al. Allow the resolving gel to polymerize in a straight line.
Carefully pour off the one al and rinse the gel with double distilled water. Pour off the water and use aous paper to absorb any leftover liquids. Quickly pour the stacking gel solution into the cassette and fill it as full as possible.
Then carefully insert a gel comb and use a big binder clip to hold the comb in place. Let the gel sit for one hour to one day To store the gel in a heat sealable pouch. Pour 20 mils of one x stacking gel buffer with 0.1%sodium azide Completely seal the pouch and make sure there are no leaks.
Store the gel at four degrees Celsius. To Prepare your protein samples, add an appropriate amount of protein into a thin walled micro fuge tube. Here we use 6.5 microliters.
Then add 7.5 microliters of no vx trix glycine SCS sample buffer. Then Add one microliter of 100 millimolar DTT, and if needed, double the distilled water for a total volume of 15 microliter To the Nature your protein. Incubate your samples at 70 degrees Celsius for 10 minutes.
Set Up the gel apparatus and pour 200 mls of one x tris glycine running gel buffer with 500 microliters of NewAge antioxidant into the inner Chamber. Then pour 300 mils of one extra glycine running buffer into the outer chamber. Carefully add your 15 microliter protein sample into a well by using a gel loading pipette tip.
When you have finished loading your samples, run the gel at 200 volts for 60 to 90 minutes. When you start your gel, the protein will start to migrate through the poly acrylamide gel based on size. Smaller proteins will run faster than larger proteins.
Carefully remove the gel from the cassette and wash the gel three times with double distilled water for five minutes with gentle agitation. After the third wash, stain the gel with in vitro gin, simply blue safe stain and gently agitate for one hour. Pour off the standing dye and detain the gel with double distilled water.
If you want shorter and darker bands, then you should run the gel for a shorter period of time. But if you want to stretch the lanes, then run it Longer To remove the epoxy so the cassettes may be reused. Use a gel knife to scrape off other epoxy.
Today we've shown you how To reuse the plastic cassettes that come with commercially available poly acrylamide gels. It's important to use epoxy rated for plastic to seal the gel cassettes. Also use high quality reagents and always wear gloves when handling un polymerized acrylamide.
Thank you and good luck with your experiments.
