How to Prepare Western Blot Transfer Buffer (Towbin)

1. 1Step 1: Measure approximately 300 mL of distilled water in a 500 mL beaker
2. 2Step 2: Add 1.51 g Tris base while stirring until completely dissolved
3. 3Step 3: Add 7.21 g glycine while stirring until completely dissolved
4. 4Step 4: Transfer solution to 500 mL volumetric flask
5. 5Step 5: Add 100 mL HPLC-grade methanol and mix thoroughly
6. 6Step 6: Fill to 500 mL mark with distilled water and mix well
7. 7Step 7: Check pH (should be ~8.3, do not adjust as this will affect protein transfer)
8. 8Step 8: Filter through 0.22 μm membrane if sterile solution needed

Filter sterilization through 0.22 μm membrane filter if sterile solution required. Do not autoclave as methanol will evaporate and concentrations will change.

Level 2 safety required. Wear nitrile gloves, safety glasses, and lab coat. Use fume hood when handling methanol. Methanol is flammable and toxic - avoid inhalation and skin contact. No ignition sources near methanol.

Store at 4°C in tightly sealed container. Can be stored at room temperature for short-term use. Methanol may evaporate over time, so replace solution if concentration accuracy is critical. Stable for 6 months when properly stored.