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