Acrylamide gels

Pouring Tris-Tricine Acrylamide Gels

Protocol by jcb

Running Gel (10%)

• 4.9 mL 30% acrylamide/0.8% bisacrylamide (37.5:1)
• 5 mL Tris-Cl/SDS pH 8.45
• 1.15 mL MilliQ water
• 3.95 mL 40% glycerol

• 25 uL 10% APS (made fresh)
• 5 uL TEMED

Stacking Gel (4%)

• 0.81 mL 30% acrylamide/0.8% bisacrylamide
• 1.55 mL Tris-Cl/SDS pH 8.45
• 3.89 mL MilliQ water
• 40 uL saturated bromophenol blue (optional)

• 37.5 uL 10% APS (0.1g ammonium persulfate/mL H20)
• 17.5 uL TEMED

Add APS and TEMED immediately before pouring.

Pour running (separating) gel (use 20G 1.5 syringe) to 1 cm below bottom of comb (mark short plate) and overlay with isopropanol. Set for 1 hr, rinse with water (buffer might be better), and pour stacking gel. Insert combs, being careful to avoid bubbles. Let set, wrap in damp paper towels, and store 4 C for up to one week.

15 well, .75 mm gels hold about 15 uL sample per lane. To run gels (MiniProtean III), put 200 mL Anode Buffer in lower/outer chamber and 125 mL Cathode Buffer in inner/upper chamber. Remove combs after adding running buffers. Use a syringe or x-long gel-loading pipette tip to flush out each well gently with Cathode Buffer to remove any unpolymerized acrylamide.

Run at about 80 V for a couple of hours.

Tris-Tricine Cathode Buffer

• 12.22 g Tris (0.1 M)
• 17.92 g Tricine (0.1 M)
• 1 g SDS (0.1%)
• to 1 L with MilliQ H2O (no need to pH)

Tris-Tricine Anode Buffer (5L)

• 121 g Tris (0.2 M)
• 500 mL MilliQ H2O
• pH to 8.9 with 6N HCl (about 30 mL)
• to 5 L with MilliQ

Tris-Cl/SDS pH 8.45

• 182 g Tris base in 300 mL MilliQ H2O (Tris is 121.14 g/mol) (hard to dissolve)
• to pH 8.45 with 3N Hcl
• MilliQ H2O to 500 mL
• Filter 0.45 um
• Add 1.5 g SDS