NuPAGE Novex Tris-Acetate Gels

Prepare Samples

Reagent Denaturing Sample* Native Sample

NuPAGE LDS Sample Buffer (4x) 2.5 ml --

Tris-Glycine Native Sample Buffer (2x) -- 5 ml

dH₂O to 7.5 ml to 5 ml

Total Volume 10 ml 10 ml

Heat Samples 70°C for 10 min. Do not heat.

*For reduced samples, add NuPAGE Reducing Agent (10x) to 1x.

Preparing 1x Running Buffer

Denaturing Samples: Add 50 ml 20x NuPAGE Tris-Acetate SDS Running Buffer to 950 ml dH₂O.

Native Samples: Add 100 ml 10x Tris-Glycine Native Running Buffer to 900 ml dH₂O.

Load Samples

Load the appropriate concentration of your protein sample on the gel.

Load Buffer

Fill Upper (200 ml) and Lower (600 ml) Buffer Chambers with the appropriate 1x Running Buffer.

For Reduced Samples: Use 200 ml 1x Running Buffer with 500 ml NuPAGE Antioxidant in the Upper Buffer Chamber.

Run Conditions

Voltage: 150 V constant

Run Time: 1 hour (denaturing gel), 2-3 hours (native gel)

Expected: 40-55 mA/gel (start); 25-40 mA/gel (end) for denaturing gel

18mA/gel (start); 7mA/gel (end) for native gel

Gel

(Hand-written annotations found on original copy).

1. Make 600 ml running buffer (H₂O in bottle, 30 ml 20x, H₂O to 600 ml mark).
2. Take out gel, remove comb and tape on bottom.
3. Put into chamber, put in 2 gel, clamp closed w/ white block/clamp device.
4. Fill upper chamber, add 0.5 ml antioxidant.
5. Rinse wells.
6. Load 7 markers, well holds ~35?

*Adr1 extracts ~10-20 ml.

7. Up to 150 V, ~ 1hr.