Using a lot of SDS-PAGE gels for Western blot and Coomassie? Precast gels are stored for too long and expired? Why not try to cast your own SDS-PAGE gels to save some budget for the lab as well as guarantee a good result of your experiment. Today, we would like to share five tips for handcasting SDS-PAGE gels, as well as the recipe and protocol.
5 Tips During Operation
Check all of the following bullets when operating. They are the key to successfully made SDS gels.
1. All Equipment meets protocol requirement
- Make sure the glass plates are clean enough.
- 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 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 done as fast as possible.
- Work in a hood while adding TEMED, given that it has a very strong smell.
3. Keep an eye on iso-propanol evaporation
- Usually the iso-propanol will run out within 2 hours. Hence, if you cannot finish casting the gels within that time frame, make sure to keep refilling the iso-propanol to prevent the resolving gel from drying.
4. APS solution needs to be freshly made
- Make fresh APS solution every day for the best performance. The accumulation of water in APS will cause a rapid loss of reactivity.
- Handcasted gels should be stored at 4°C for up to one week.
- We don’t suggest long-term storage of the handcasted gel (more than one week), as it may affect subsequent running 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 comb, with or without stacking gel, etc.).
|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|
|40% Acrylamide/Bis||0.50 ml|
|0.5M Tris pH 6.8||1.26 ml|
|10% SDS||3.18 ml|
|10% APS||25 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 made)
Deionized water (dI-H2O)
Mini-PROTEAN® Tetra Cell Casting Module (BIO-RAD): casting stands with gaskets, casting frames, combs, short plates and spacer plates.
15mL Falcon tube
- 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), add reagents except for TEMED respectively in a 15mL Falcon tube. Before adding TEMED, vortex the tube and make sure the solution is well mixed.
- Add TEMED to the tube, use the serological pipette to mix the solution quickly.
- Use 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 till full.
- Insert the comb and wipe the overflowing solution. Allow the the gel to polymerize for 20-30min or until a line is seen between the stacking and resolving gels.
- Remove the comb by pulling it straight up slowly and gently. Rinse the wells completely with deionized water.