A Complete Guide to Handcasting SDS-PAGE Gels

Lingyi Tong

Lingyi Tong
Feb 28, 2020 4:43:07 PM

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.

5. Storage

  • 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.).

Table 1. Resolving Gel Solution
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
Table 2. Stacking Gel Solution (2 Gels)
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 made)

Tetramethylethylenediamine (TEMED)

Isopropanol

Deionized water (dI-H2O)

70% Ethanol

Mini-PROTEAN® Tetra Cell Casting Module (BIO-RAD): casting stands with gaskets, casting frames, combs, short plates and spacer plates.

15mL Falcon tube

Pipette

Serological pipette

 

Protocol

  1. Clean and wipe the glass plates with 70% ethanol.
  2. Set up the cell casting module, making sure the glass plates are held tightly.
  3. 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.
  4. Add TEMED to the tube, use the serological pipette to mix the solution quickly.
  5. 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.
  6. Fill the space with isopropanol, then wait for 30min until the gel is polymerized.
  7. Discard the isopropanol and rinse with deionized water.
  8. Discard deionized water.
  9. Follow the stacking gel recipe (see Table 2) to prepare the stacking gel solution, following the same procedure as step 3.
  10. Cast the stacking gel solution into the space between the two glass plates till full.
  11. 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.
  12. Remove the comb by pulling it straight up slowly and gently. Rinse the wells completely with deionized water.

 

 

Tags: Western Blot, SDS-PAGE, Handcast Gel

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