Peptide-Carrier Kit Protocol II: Coomassie Blue SDS-PAGE
This kit provides carrier protein (0.2 mg/ml) for 50 ligation reactions (at least 250 lanes) for Western blot analysis (Protocol I). The extent of ligation is typically 75-90% when a final peptide concentration of 0.5 mM is used. It is not necessary to run Coomassie Blue stained SDS-PAGE unless you wish to check the extent of ligation.
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.
Note: Please refer to Table 1 for a list of reagents that may affect the extent of ligation.
- 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.