Using the NuPAGE Bis-Tris Mini Gel for protein purification experiments on transgenic Nemtostella vectensis embryos.

Here I used the manufacturer protocol with some modifications.

Prepare samples

Volumes are provided for a 10-μL sample size.

Components Reduced sample Non-reduced Sample
Sample x μL x μL
NuPAGE™ LDS Sample Buffer (4X) 2.5 μL 2.5 μL
Reducing Agent (2-mercaptoethanol) 2.5% final concentration —
Deionized Water to 6.5 µL to 7.5 µL
Total Volume 10 µL 10 µL

Heat samples at 70˚C for 10 minutes.

Prepare buffers

Add 50 mL of 20X NuPAGE MES SDS Running Buffer to 950 mL of deionized water to prepare 1X SDS Running Buffer.

Prepare gel

  1. Remove the comb, and rinse the gel wells three times using 1X Running Buffer.
  2. Remove the white tape near the bottom of the gel cassettes.
  3. Place the gels in the mini gel tank

Load buffers

For the XCell SureLock™ Mini-Cell, add 600 mL of 1X running buffer to the lower chamber, and 200 mL to the upper chamber.

Load samples and molecular marker

  1. Load 10 µL of your samples in the appropriate wells.
  2. Load your protein ladder in the appropriate well.

Run the gel

Run for 25 minutes at 200 V constant.

Gel staining

I used the AcquaStain Protein Gel Stain.

  1. Add enough stain to cover gel completely.
  2. Incubate overnight at 4°C.
  3. Wash the gel in deionized water for at least 20 min.