SDS-PAGE Gel Preparation and Running (Laemmli discontinuous buffer system)

• Acrylamide / bisacrylamide (unpolymerised). NEUROTOXIN. Wear gloves, eye protection, lab coat. Never mouth-pipette. Handle only in a dedicated acrylamide-designated area. Polymerised gels (the actual running gels) are safe.
• TEMED. Pungent, flammable, irritant. Work in a fume hood or well-ventilated area. Gloves.
• APS (ammonium persulfate). Strong oxidiser; irritant. Standard gloves and goggles. Fresh solution every 1–2 weeks (degrades).
• SDS. Respiratory irritant as powder; handle dry SDS in a fume hood. Solutions safe.
• β-mercaptoethanol. Strong odour; toxic if ingested. Use in a fume hood; gloves + eye protection.
• Coomassie stain. Low acute toxicity but stains everything — wear lab coat.
• Electric hazard. Gel electrophoresis runs at 100–300 V; never touch the apparatus while powered on. Power off before opening.
• Hot samples. Sample buffer heat-treated at 95°C; brief burns risk when handling tubes out of heat block.

Buffer preparation (prepare in advance):

• Resolving gel buffer — 1.5 M Tris-HCl pH 8.8. Store room temperature; stable for months.
• Stacking gel buffer — 1.0 M Tris-HCl pH 6.8. Store room temperature; stable.
• Running buffer (1X TGS) — 25 mM Tris base + 192 mM glycine + 0.1% SDS. Prepare 10X stock; dilute fresh for each run.
• Laemmli sample buffer (2X) — 100 mM Tris-HCl pH 6.8 + 4% SDS + 20% glycerol + 10% β-mercaptoethanol + 0.1% bromophenol blue. Aliquot and store −20°C; stable for 6+ months.
• 10% APS — 10% w/v ammonium persulfate in water. Prepare fresh weekly (degrades).
• 30% acrylamide/bisacrylamide (29:1) — commercial solution (Bio-Rad, Invitrogen). Store 4°C, protected from light. Do not use once cloudy.

Gel recipe (per one 8 × 10 cm × 1 mm gel):

Resolving gel (10% for proteins 15–100 kDa; adjust for smaller/larger targets):

• 4 mL 30% acrylamide/bis (29:1)
• 3 mL resolving gel buffer (1.5 M Tris pH 8.8)
• 4.9 mL water
• 120 µL 10% SDS
• 120 µL 10% APS
• 12 µL TEMED

Stacking gel (4%):

• 0.65 mL 30% acrylamide/bis
• 1.25 mL stacking gel buffer (1 M Tris pH 6.8)
• 3.05 mL water
• 50 µL 10% SDS
• 50 µL 10% APS
• 5 µL TEMED

Equipment:

• Vertical gel casting stand and plates (1 mm spacers).
• Tank + lid with electrodes.
• Power supply capable of 200 V.
• Comb (10-well or 15-well).
• Heat block for sample denaturation.

Mental model: SDS coats denatured proteins uniformly, giving them a mass-proportional negative charge. In an electric field, proteins migrate toward the anode at a rate inversely proportional to their size — smaller proteins move faster. The discontinuous buffer system (different pH in stacking vs resolving) concentrates proteins into a sharp band before separation, giving crisp resolution.

Percentage-choice guide:

• 8% gel: 50–200 kDa proteins
• 10% gel: 15–100 kDa (most common — use unless you have specific range needs)
• 12% gel: 10–50 kDa
• 15% gel: small peptides (5–30 kDa)

• Gel casting (30–45 min): mix → pour resolving gel → overlay with isopropanol (20 min polymerise) → pour stacking gel + comb (20 min polymerise).
• Sample prep (10 min): mix with 2X sample buffer, heat 95°C × 5 min.
• Loading (5 min): load samples + ladder into wells.
• Running (60–75 min): 100 V × 15 min (stacking) then 150 V × 45–60 min (resolving) or until dye front reaches bottom.
• Staining (60–90 min): Coomassie stain 30 min + destain 30–60 min with fresh destain changes.
• Imaging (5 min): visible-light gel doc or phone photo on a light source.
• Total hands-on: ~90 min; hands-off polymerisation + running: ~2 hours.