How to separate proteins using an Invitrogen precast SDS-PAGE gel

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"The first step in many protein analytical methods, including western blot analysis, is to separate proteins by gel electrophoresis. The most widely used protein electrophoretic technique is SDS-PAGE, in which proteins are denatured and evenly charged by an ionic detergent so that protein migration through the gel is based only on their size. Thermo Fisher Scientific offers a wide variety of Invitrogen precast gels in different chemistries, percentages, gradients, and sample well configurations. Make sure to choose the right protein gel based on the size of your protein and your downstream applications. This video will show you how to separate protein by using Invitrogen Mini Gel Tank, which is compatible with a wide range of precast and pour-your-own gels and the Invitrogen Bolt Bis-Tris Plus precast gel, with a wedge-shaped well designed to enable the loading of sample volumes up to 60 µL per well. To replicate what you see in this video you will need Bolt LDS Sample Buffer, a Bolt Mini Gel, Bolt MES SDS Running Buffer, Bolt Antioxidant, an Mini Gel Tank, a PageRuler Plus Prestained Ladder, a PowerEase Power Supply, and a gel knife. Inspect the Mini Gel Tank by making sure that the electrophoresis tank is secure in the stand and that the cassette clamps are in place. The anode connectors should be positioned in the middle of the tank. The fill line that is on the front of the tank should be facing you. Partially prefill the tank chambers that will be used by adding 1X running buffer until it reaches the cathode. Once the tank is set up, cut open a gel cassette pouch and remove the cassette from the pouch. Gently remove the comb either by sliding the comb up one side at a time or by using both thumbs to push both sides of the comb up simultaneously. Wash the cassette wells with running buffer and invert to remove any excess. Do this two more times. Make sure you remove the tape at the bottom of the cassette. In order to make the gel easier to load, place the gel cassettes in front of the cassette clamps in the tank with the wells facing toward you- hold the gel so that it doesn’t drop down all the way in the tank while pulling the cam handle forward. This will hold the gel firmly in place while you load samples and standards. If you’re running two gels, repeat this for the other gel. Fill the wells of the gel with running buffer. Simply lower the pipette tip into the well and pipette the sample into the well. The front-loading, wedge-shaped wells make it easy to load samples using a standard pipette tip. Load the Prestained Ladder into a separate well in order to assess the relative molecular weight of the target proteins and monitor the separation during electrophoresis. After loading the samples and standards, carefully release the cassette clamp while holding the loaded gel. To prevent disturbing the loaded samples and standards, gently slide the gel down so that it rests on the bottom of the tank. Next, pull the cassette clamp forward. With the gel cassette and clamp secured, fill the 2 chambers of the electrophoresis tank with running buffer to the fill line. It is normal for buffer to overflow to outer chambers. For reduced samples, add 400 microliters of Bolt antioxidant to the cathode buffer chamber for better protein resolution. Place the lid on top of the tank and align the electrode receptacles in the lid with the corresponding electrodes in the tank. Then push the lid down firmly. Connect the electrode cords to the power supply. Set the power supply to the appropriate run voltage and time. Bolt gels can be run at 200 volts and electrophoresis can be complete in as little as 20 minutes. Now, start the run. Bubbles appearing around the cathode indicate that the current is flowing. At the end of the run, as determined by time or when the dye front reaches the foot of the gel, turn off the power supply, disconnect the cords, and remove the lid by placing your thumb on the tank handles and pulling the lid up with your fingers. Remove the gel cassette from the electrophoresis tank by releasing the cam handle. Carefully separate the gel from the plates using the gel knife. Remove the foot of the gel and wells. The gel is now ready for western blot transfer, Coomassie stain, or another protein analysis method. Find out more information about Invitrogen precast gels at thermofisher.com/protein gels

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