SDS-PAGE Using TGX Acrylamide Kit--CHEM 584

Assemble the gel cassette on the casting stand. We are pouring a 0.75 mm thick gel and the volumes in this protocol are correct for this thickness of the gel.

Watch this YouTube video to familiarize yourself with how to assemble and pour your gel. TGX Stain-Free™ FastCast™ Acrylamide Gel Casting - YouTube

Prepare the resolving gel acrylamide solution by combining 2 mL of Resolver A & B solutions in a 15 mL tube.

Add 2 µL of TEMED and 20 µL of freshly prepared 10% w/v APS to the combined resolver solution and mix well. Use an appropriate pipette to steadily dispense the solution into the prepared gel cassette. Do not let bubbles form of solution mix with air. Fill the cassette to 0.5-1.0 cm below the bottom of the teeth on the comb. Immediately prepare and pour the stacking gel solutions as directed in the next steps.

Prepare the stacking gel acrylamide solution by combining 1 mL of the stacker A & B solutions in a 15 mL tube.

Add 2 µL of TEMED and 10 µL of the freshly prepared 10% w/v APS to the combined stacker solution and mix well. Pipet solution down the middle of the gel cassette, filling to the top of the short plate. Apply slowly and steadily to prevent mixing with the resolving solution. Align and insert the comb in the cassette carefully to prevent air from being trapped under the comb teeth. Allow the gel to polymerize for 30-45 min before electrophoresis.

Use Laemmli sample buffer (4x) to prepare your samples. Your sample wells should hold up to 33 µL. You do not need to add sample buffer to the protein ladder, you just add the 5 µL aliquot of the protein ladder directly to a lane on your gel.

Load your gel into the gel box. Here is a YouTube link to watch the process. Quick Tips: How to Load a Mini-PROTEAN TGX Stain-Free Gel for Electrophoresis - YouTube There are new and old gel boxes, so if you are using an older model, you can ask the TA or the Instructor for assistance.

Fill the inner chamber completely with 1x Running Buffer and the outer chamber to the line for the number of gels you are planning to run.

Run your gel between 100-200 V or until the loading dye has just run off the bottom of the gel.