Our protocol demonstrates how to pour multiple protein gels at a time by recycling Invitrogen Nupage Novex minigel cassettes, and inexpensive materials purchased at a home improvement store. This economical and streamlined method includes a way to store the gels at 4°C for a few weeks. By re-using the plastic gel cassettes from commercially available gels, labs that run frequent protein gels can save significant costs and help the environment.
The evaluation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is a common technique used by biochemistry and molecular biology researchers1-4. For laboratories that perform daily analyses of proteins, the cost of commercially available polyacrylamide gels (˜$10/gel) can be considerable over time. To mitigate this cost, some researchers prepare their own polyacrylamide gels. Traditional methods of pouring these gels typically utilize specialized equipment and glass gel plates that can be expensive and preclude pouring many gels and storing them for future use. Furthermore, handling of glass plates during cleaning or gel pouring can result in accidental breakage creating a safety hazard, which may preclude their use in undergraduate laboratory classes. Our protocol demonstrates how to pour multiple protein gels simultaneously by recycling Invitrogen Nupage Novex minigel cassettes, and inexpensive materials purchased at a home improvement store. This economical and streamlined method includes a way to store the gels at 4°C for a few weeks. By re-using the plastic gel cassettes from commercially available gels, labs that run frequent protein gels can save significant costs and help the environment. In addition, plastic gel cassettes are extremely resistant to breakage, which makes them ideal for undergraduate laboratory classrooms.
1. Preparing the cassettes
2. Pouring the resolving gel
3. Pouring the stacking gel
4. Storing the gel
5. Preparing the Samples
6. Running the gel
7. Staining the gel
8. Cleaning the cassettes
9. Representative Results
Because epoxy will form a water-tight seal, (Figure 1), the cassettes will not leak when the acrylamide solution is poured into them. In addition, the gel cassettes can be easily opened to retrieve the gel for staining, and the epoxy can be peeled off of the cassettes to allow their continued re-use. As shown in Figure 2, the gel shows clear separation of protein markers and sample bands suitable for routine protein electrophoresis.
Figure 1. Application of epoxy to seal minigel cassettes. A thin layer of epoxy is applied to the edge of one of the cassettes prior to assembly. To highlight the epoxy, it was false-colored red in this image.
Figure 2. Separation of proteins on an SDS-Tris-glycine gel poured using re-used minigel cassettes. SDS-PAGE was performed as described above. Following electrophoresis the gel was removed from the minigel cassette, stained in SimplyBlue SafeStain as per manufacturer’s instructions, and photographed using transmitted white light. Lane M contains SeeBlue Plus2 pre-stained protein standards. The molecular weights of the proteins in lane M are indicated on the right side of the gel in kilodaltons (kDa). The other lanes show proteins from a cleared lysate from bacteria expressing a glutathione-S-transferase (GST)-tagged protein. The proteins shown are those washed from the glutathione beads prior to elution of the GST-tagged protein.
Protein gel electrophoresis is an indispensible method for most molecular biology laboratories. Commercially available minigels provide a high level of convenience, but can be costly. Here we show how to re-use the minigel cassettes from a popular brand of commercially available minigels. This method retains the convenience of having a ready-made source of gels, but at a fraction of the cost of commercially available minigels. A key step in this procedure is applying the correct amount of epoxy to completely seal the cassettes prior to adding the unpolymerized acrylamide to prevent leakage. Use only epoxy that is rated for plastic, and allow enough time for polymerization of the epoxy as described in the manufacturer’s instructions. For added convenience, multiple cassettes can be sealed and stored prior to pouring the gels. When pouring the resolving gel, be sure to leave enough room for the stacking gel, typically about 0.5 cm more than the height of the comb used to form the wells. During polymerization of the resolving gel, keep the gel upright such that the buffer-saturated 1-butanol will form a straight, flat surface to form on the top of the resolving gel. If the gel does not polymerize within 30 min, the amount of 10% APS and TEMED may be increased. Once the gel has finished running, it is important to carefully break the epoxy seal to prevent ripping the thin gel. The method reported here of re-using commercial gel cassettes can save significant costs associated with frequent SDS-PAGE use. The same cassettes may be reused for years and we have no evidence that repeated use of the same cassette reduces their usability. In addition, multiple gels can be prepared and stored for weeks in a polyester barrier film bag containing a small amount of 1X stacking gel buffer containing 0.1% sodium azide.
Abbreviations:
Dithiothreitol (DTT ), N, N, N’, N’,-tetramethylethylenediamine (TEMED), Type I reagent grade water (IRG-H2O), ammounium persulfate (APS), sodium dodecyl sulfate (SDS), Tris (hydroxymethyl) aminomethane (Tris-base), N,N’-methylene-bis-acrylamide (bis-acrylamide)
The authors have nothing to disclose.
The authors thank the Howard Hughes Medical Institute for a UF Science for Life award to A.H.
Name of reagent | Company | Catalogue number | Comments |
Invitrogen Nupage Novex Minigel Cassette (1.0 mm) | Invitrogen | NC2010 | Available in 2, 5, 10, 12, 15 and 2D wells |
Plastic Epoxy | Loctite | 108771 | |
Electrical Tape | Scotch | ||
125 mL side arm flask | Pyrex | 5360 | |
4X Tris-Base resolving gel buffer | 1.5 M Tris-base pH 8.7 | ||
acrylamide | Fisher Scientific | 01065-500 | |
Bis-acrylamide | MP Biomedicals, Inc. | 800173 | |
30.8% acrylamide solution | 30:0.8 acrylamide: bis-acrylamide in IRG-H2O | ||
10% APS | Fisher Scientific | BP179-25 | 0.1 g ammonium persulfate in 1 ml IRG-H2O, prepared immediately before use |
TEMED | Arcos Organics | 138450500 | |
1-butanol saturated with 1X resolving gel buffer | 5:2 1-butanol to 1X resolving gel buffer | ||
4X Tris-Base stacking gel buffer | 1.5 M Tris-base pH 6.8 | ||
Gel comb | Invitrogen | NC3015 | 15 well, 1.0 mm thickness |
Impulse Sealer | TEW Electric Heating Equipment | ||
Heat Sealable Pouch | Kapak/Scotchpak | 1 pint size (6.5″ x 8″) | |
Gel storage buffer | 1X stacking gel buffer with 0.1% sodium azide | ||
Novex SDS Tris-Glycine sample buffer (2X) | Invitrogen | LC2676 | |
DTT | FisherBiotech | BP172-5 | |
10X Tris-Gylcine Running buffer | Invitrogen | LC2675 | 1L of 10X stock: 29.0 g Tris-Base, 144.0 g Glycine, 10.0 g SDS |
XCell SureLock Mini-Cell | Invitrogen | EI0001 | |
Gel Knife | Invitrogen | EI9010 | |
SimplyBlue Safe Stain | Invitrogen | LC6065 |