Control Peptide PB1 Protocol 2: Analysis of Ligation by Coomassie Blue SDS-PAGE

Introduction

We recommend the use of a Coomassie stained SDS-PAGE to check your ligated product (by a shift in mobility) only if you suspect that the ligation is not working.

Protocol

1. Dissolve your peptide in water to a concentration of 1 mM.
2. Thaw the carrier protein at room temperature.
3. Mix the following (in order):
   
4. Incubate at room temperature for 15-60 minutes or at 4°C overnight.
5. Add 12.5 µl of 3X SDS Sample Buffer.
6. Heat at 95°C for 5 minutes.
7. Load 15 µl/lane (~ 0.8 µg carrier protein) for Coomassie Blue staining. Dilute 10-20 fold for Western blot analysis.