This is an instructional video on how to prepare and store an SDS-PAGE gel. The following are all the materials, chemicals and instruments used in this experiment. Please keep in mind that acrylamide is a cancerogenic compound,

so use nitrile gloves and keep a clean surface when working with the chemical. The recipe depends on the gel you want to prepare. For specific ratios and volumes, consult with your lab supervisor. The buffers and the chemicals used are listed on the screen. In this case, we prepared two gels at the same time.

Start by setting up the casting stand and arranging the glass plates. Stack the plates with the shorter side facing towards you and carefully insert them into the frame. Once in place, secure the plates as instructed and check the alignment by running your finger along the edges.

If the alignment is correct, proceed to place the frame onto the stand. A properly secured setup should prevent any mixture from leaking out of the bottom. To confirm this, you can conduct a water test by adding a few milliliters of distilled water between the plates and observing for any signs of leakage. Remember to remove the water using filtered paper to avoid disturbing the gel ratios.

As for the resolving gel and the stacking gel, they are prepared in 50 milliliter falcon tubes prior to transferring to the cassette. First, prepare the resolving gel. Start by adding water to the falcon tube.

Next, add tris buffer. Keep in mind not to mix the tris buffers used since each gel is prepared with the buffer of a different pH level. For the resolving gel, add 1.5 molar tris buffer at a pH of 8.8.

Carefully pipette the acrylamide solution into the falcon tube.

To achieve that constant mass-to-charge ratio of proteins, add the anionic detergent SDS.

Before adding the last two components, keep in mind that ammonium persulfate or APS is an oxidizing agent used with TEMED to catalyze the polymerization. Once both are added to the mixture, the gel begins polymerizing. Here, TEMED is added first without APS. Don't forget to mix by twirling the tube.

Finally, APS is added to catalyze the polymerization process.

Immediately after, the mixture is transferred to the cassette using a pipette. Add until about 1 cm below the top so that the comb does not reach the resolving gel later on. Here, we have marked this point with a marker on the frame.

Immediately after, add isopropanol to remove any air bubbles and smoothen the surface of the gel. Allow the gel to polymerize. In the meanwhile, begin preparing the stacking gel.

To prepare the stacking gel, the same procedure is carried out as before, except that here we use the Tris buffer with a pH of 6.8. Remember not to add APS after adding TEMED to the solution until the gel is ready to be transferred.

After 20 to 30 minutes, the resolving gel is ready. You can confirm using the amount left in the Falcon tube as seen in the video. Remove the isopropanol by inverting the frame.

Rinse with distilled water a couple times and place back in the stand. Add APS to the stacking gel and transfer immediately on top of the resolving gel.

Instead of using isopropanol to avoid the air bubbles, we insert the comb with the number of wells we need. To prevent trapping of air bubbles under the comb, fill the plates up to the brim. Allow the gel to polymerize for 20 to 30 minutes.

After polymerization of the stacking gel, the SDS paste gel can be stored in the fridge at 4°C for a few days up to a week. However, this remains to be discussed with your lab supervisor. Remove the frame and, keeping the plates and comb intact, wrap the gel in tissues.

Make sure not to move the comb, otherwise the wells will be damaged.

Add distilled water to the tissues and store in a plastic bag to trap the moisture. Don't forget to label the bag using your initials and the date of when the gel was prepared. This way, you have managed to successfully prepare and store an SDS paste gel.