Protein analysis: SDS-PAGE
Principle of SDS-PAGE analysis
Preparation of dilute or salt-containing samples for SDS-PAGE
Separation of proteins by SDS-PAGE
- Assemble gel plates with spacers according to the manufacturer’s instructions.
Tip: The plates should be thoroughly cleaned and dried before use.
- Mark the level to which the separating gel should be poured — a few millimeters below the level where the wells will be formed by the comb.
- Mix the following in a beaker or similar vessel (for a 12% acrylamide 8 x 8 or 8 x 10 cm, 1 mm thick, minigel).
2.2 ml 30% acrylamide/0.8% bis-acrylamide stock solution
2.2 ml 2.5x separating gel buffer
1.1 ml distilled water
5 µl TEMED
The volumes of acrylamide/bis-acrylamide solution and water should be adjusted according to the percentage acrylamide required (dependent on the size of protein to be separated; see table Compositions and separation properties of SDS-PAGE gels).
Tip: The size of the gel apparatus used will determine the volumes of gel solutions necessary.
- Just before pouring, add 50 µl 10% ammonium persulfate, and mix well. Pour the gel between the assembled gel plates to the level marked in step 2. Overlay with butanol.
Tip: Water can be used instead of butanol when using apparatus that may be damaged by the use of butanol — see the manufacturer’s instructions.
Tip: As soon as ammonium persulfate is added, the gel should be poured quickly before the acrylamide polymerizes.
Tip: Prepare ammonium persulfate solution freshly each time it is required
- After polymerization is complete (around 20 min), pour off butanol, rinse with water and dry.
Tip: Water remaining on the plates can be removed using pieces of filter paper.
- For the stacking gel, mix the following:
0.28 ml 30% acrylamide/0.8% bis-acrylamide stock solution
0.33 ml 5x stacking gel buffer
1 ml distilled water
2 µl TEMED
- Just before pouring, add 15 µl 10% ammonium persulfate, and mix well. Pour on top of the separating gel. Insert comb, avoiding introduction of air bubbles.
Tip: As soon as ammonium persulfate is added the stacking gel should be poured quickly, before the acrylamide polymerizes.
Tip: With a marker pen, mark and/or number the positions of the wells before removing the comb. This aids loading of samples.
- After the stacking gel polymerizes (around 10 min), the gel can be placed in the electrophoresis chamber. Fill the chamber with electrophoresis buffer and remove the comb.
- Before loading, add 1 volume 5x SDS-PAGE sample buffer to 4 volumes of protein sample (i.e., add 2 µl sample buffer to 8 µl sample giving a final volume of 10 µl). Vortex briefly and heat at 95°C for 5 min.
Tip: During heating at 95°C, release pressure build up in tubes by briefly opening lids, or piercing a small hole in the lid with a needle. After heating, samples should be briefly centrifuged and vortexed.
- Load samples and run gel. For electrophoresis conditions refer to the recommendations provided by the manufacturer of the apparatus.
Tip: Before loading the samples, rinse out wells with 1x electrophoresis buffer using a suitable syringe and needle.
Tip: Load empty wells with 1x SDS-PAGE sample buffer to ensure that sample lanes do not spread out. Tip: Ensure that the electrodes are correctly connected. The proteins will migrate towards the positive (labeled +, usually red) electrode.
Tip: Running the gel until the bromophenol blue dye reaches the bottom edge usually gives a satisfactory spread of protein bands.
Visualization of proteins in SDS-PAGE gels
- Incubate the gel in Coomassie staining solution for between 30 min and 2 h with gentle shaking.
Tip: Coomassie Brilliant Blue R reacts nonspecifically with proteins.
- Gently agitate the stained gel in destaining solution until the background becomes clear (1–2 h).
Tip: A folded paper towel placed in the destaining bath will soak up excess stain and allow the reuse of destaining solution.
After destaining the proteins appear as blue bands against a clear gel background. Typically, bands containing 50 ng protein can be visualized.