How to prepare protein interaction partners for MS analysis
Immunoprecipitation (IP) followed by mass spectrometry (MS) analysis is a powerful method to identify interaction partners of a protein of interest. However, sometimes it can be difficult to obtain reliable results.
Now, the combined use of ChromoTek GFP-Trap for immunoprecipitation and PreOmics iST sample preparation kit is in fact a straight forward, reproducible, and reliable approach to identify protein interaction partners. It even preserves posttranslational modification (PTM) depending protein-protein interactions.
In a recent applications note we have demonstrated how protein interaction partners of PARP1 can be identified: We have immunoprecipitated eGFP-tagged PARP1 protein and its interacting partners using the GFP-Trap® Magnetic Particles M-270 and subsequently processed the pulldown for MS analysis following the instructions of the iST kit.
PARylation mediates the interaction of PARP1 to several DNA repair proteins. Our results show that those interactions are not disrupted by neither the IP nor the MS sample preparation used herein. Therefore, the streamlined combination of the GFP- Trap and the iST kit’s workflow proves to preserve PTM-mediated protein-protein interactions.
The PreOmics iST kit can also be used with other ChromoTek Nano-Traps:
- ChromoTek iST GFP-Trap Kit for AP-MS sample preparation of GFP-fusion proteins
- ChromoTek iST mNeonGreen-Trap Kit for AP-MS sample preparation of mNeonGreen-fusion proteins
- ChromoTek iST Myc-Trap Kit for AP-MS sample preparation of Myc-tagged proteins
- ChromoTek iST RFP-Trap Kit for AP-MS sample preparation of RFP-fusion proteins
- ChromoTek iST Spot-Trap Kit for AP-MS sample preparation of Spot-tagged proteins
- ChromoTek iST TurboGFP-Trap Kit for AP-MS sample preparation of TurboGFP-fusion proteins
- ChromoTek iST V5-Trap Kit for AP-MS sample preparation of V5-tagged proteins