2X Laemmli sample buffer recipe
SDS-PAGE sample buffer mixed 1:1 with samples: 125 mM Tris-HCl pH 6.8, 4% SDS, 20% glycerol, 10% β-mercaptoethanol, 0.004% bromophenol blue.
🧮 Volume calculator
| Component | Amount | Final concentration (1X) |
|---|---|---|
| 1 M Tris-HCl (pH 6.8) | 1.25 mL | 125 mM |
| SDS | 400 mg | 4% |
| Glycerol | 2 mL | 20% |
| Bromophenol blue | 0.4 mg | 0.004% |
| β-mercaptoethanol (add fresh) | 1 mL | 10% |
| Water (distilled) | to final volume |
Steps
- Mix everything except β-ME and make up to 90% of the final volume.
- Aliquot and store at room temperature or −20 °C.
- Just before use add 100 µL β-ME per 900 µL (or DTT to 100 mM).
- Mix 1:1 with sample and heat at 95 °C for 5 min.
Good to know
- Weighing 0.4 mg of bromophenol blue is hard — add 40 µL of a 1% solution instead.
- Use 4X when samples are dilute, to save loading volume.
⚠ Safety β-mercaptoethanol is toxic — handle in a fume hood.
FAQ
Should membrane proteins be boiled?
Multi-pass membrane proteins can aggregate when boiled; use 37 °C for 30 min or 70 °C for 10 min.
This is a widely used standard composition. Amounts depend on the hydrate form and purity of your reagents — check the formula weight (FW) on the bottle and follow your institution's safety rules and the manufacturers' instructions.