Do you find yourself using a lot of SDS-PAGE gels for Western blotting and Coomassie staining? Or have your pre-cast gels been stored for too long and expired? Why not try to cast your own SDS-PAGE gels to save some budget for the lab, and produce just as valid of results. Today, we would like to share five tips for hand-casting SDS-PAGE gels, as well as the protocol and formulation to do so.
5 Tips During Operation
Be sure to check all of the following bullet points when operating. They are key to successfully produce SDS gels.
1. All equipment meets protocol requirements
- Make sure the glass plates are thoroughly cleaned.
- Double-check whether the glass plates are held very tightly to avoid leaking when setting up the cell casting module.
- Make sure the size of the combs matches the spacer plates.
2. Notes for TEMED
- While making gel solution, TEMED has to be added last since it will immediately start to react with APS, and catalyze the polymerization of acrylamide and bisacrylamide. As a consequence, the following mixing and casting steps have to be completed as quickly as possible.
- Be sure to work under a fume hood while adding TEMED. It has a very strong odor!
3. Keep an eye on isopropanol evaporation
- Usually, isopropanol will fully evaporate within 2 hours. Hence, if you cannot finish casting the gels within that time frame, be sure to keep refilling the isopropanol to prevent the resolving gel from drying.
4. APS solution must be freshly prepared
- Prepare fresh APS solution every day for best performance. The accumulation of water in APS, as can happen with an older solution, will cause a rapid loss of reactivity.
5. Storage
- Hand cast gels should be stored at 4°C for up to one week.
- We don’t suggest long-term storage of the hand-casted gel (ie. for more than one week), as it may deteriorate over that time and affect subsequent experimental results.
Recipes for Gel Solution
Table 1 and Table 2 show our suggested volumes for making resolving gel and stacking gel. Volumes listed in Table 1 are required to completely fill a gel cassette, and Table 2 is for 2 stacking gels. Amounts may be adjusted depending on the application (with or without the comb, with or without stacking gel, etc.).
| Reagents | 8% | 10% | 12% | x% |
| 40% Acrylamide/Bis | 2 ml | 2.5 ml | 3 ml | 0.25x ml |
| 1.5M Tris pH 8.8 | 2.5 ml | 2.5 ml | 2.5 ml | 2.5 ml |
| dI-H2O | 5.35 ml | 4.85 ml | 4.35 ml | 7.35-0.25x ml |
| 10% SDS | 100 µl | 100 µl | 100 µl | 100 µl |
| 10% APS | 50 µl | 50 µl | 50 µl | 50 µl |
| TEMED | 5 µl | 5 µl | 5 µl | 5 µl |
| Reagents | |
| 40% Acrylamide/Bis | 0.50 ml |
| 0.5M Tris pH 6.8 | 1.26 ml |
| dI-H2O | 50 µl |
| 10% SDS | 3.18 ml |
| 10% APS | 25 µl |
| TEMED | 5 µl |
Reagents and Module
- 40% Acrylamide/Bis Solution
- 1.5M Tris pH 8.8
- 0.5M Tris pH 6.8
- 10% Sodium Dodecyl Sulfate (SDS)
- 10% Ammonium Persulfate Solution (APS, freshly prepared)
- Tetramethylethylenediamine (TEMED)
- Isopropanol
- Deionized Water (diH2O)
- 70% Ethanol
- Mini-PROTEAN® Tetra Cell Casting Module (BIO-RAD): casting stands with gaskets, casting frames, combs, short plates, and spacer plates.
- 15 mL Falcon Tube
- Micropipette
- Serological Pipette
Protocol
- Clean and wipe the glass plates with 70% ethanol.
- Set up the cell casting module, making sure the glass plates are held tightly.
- Follow the resolving gel recipe (see Table 1 above), add reagents except for TEMED to a 15mL Falcon tube. Before adding TEMED, vortex the tube and make sure the solution is well-mixed.
- Add TEMED to the tube, then use a serological pipette to mix the solution quickly.
- Use a pipette to transfer the solution into the space between the two glass plates, until it reaches the middle of the top green bar of the casting frames.
- Fill the space with isopropanol, then wait for 30min until the gel is polymerized.
- Discard the isopropanol and rinse with deionized water.
- Discard deionized water.
- Follow the stacking gel recipe (see Table 2) to prepare the stacking gel solution, following the same procedure as step 3.
- Cast the stacking gel solution into the space between the two glass plates.
- Insert the comb and wipe the overflowing solution. Allow the gel to polymerize for an additional 20-30 min, or until a line becomes visible between the stacking and resolving gels.
- Remove the comb by pulling it straight up, slowly, and gently. Rinse the wells completely with deionized water.
If you enjoyed these helpful tips for hand-casting SDS-PAGE gels, check out our curated list of blogs here to learn more about other popular techniques, and experimental troubleshooting.
