GFP-Trap®Multiwell Plate for convenient high throughput analysis of protein interactions. It consists of our anti-GFP VHH immobilized in multiwell plates. The GFP-Trap Multiwell Plate is optimized for immunoprecipitation (IP), IP-MS, and ELISA. Here you can find the protocol for sandwich ELISA und immunoprecipitation (IP) with GFP-Trap
The pull-down of proteins is an established technology. It can be difficult, particularly when the protein of interest is expressed at low levels. Here we discuss 5 tips that help you to improve your IP results.
ChromoTek GFP-Trap® is optimized for the immunoprecipitation of GFP-tagged proteins and their interacting factors.
The GFP-Trap consists of the ChromoTek anti-GFP Nanobody/ VHH that is coupled to 3 different types of beads or is immobilized in a 96 multiwell plate:
- Agarose beads
- Magnetic Agarose beads
- 96 Multiwell Plate
Immunoprecipitation with GFP-Trap.
I: Input, FT: Flow-Through, B: Bound
Based on the properties of the different matrices (see table), we recommend:
GFP-Trap® Dynabeads is optimized for the immunoprecipitation (IP) of large GFP-tagged proteins and for Co-IP of protein complexes. It consists of ChromoTek’s established anti-GFP Nanobody conjugated to Dynabeads™. Hence, also GFP-Trap Dynabeads has the very high affinity of 1 pM like GFP-Trap Agarose and Magnetic Agarose.
Jellyfish Green Fluorescent Protein (GFP) and its derivatives are still the most frequently used fluorescent proteins in biomedical research. Recently, additional green fluorescent proteins have been discovered in higher animals such as crustaceans and lancelets. These FPs share a common fold, but diverge widely in their primary sequence. Thus, they require novel, dedicated antibody research tools. Here is an overview about EGFP (the most commonly used GFP derivative), TurboGFP and mNeonGreen.
What makes on-bead digestion favorable?
Just pull down your protein of interest with immobilized nanobodies, also termed VHHs or single domain antibodies. Then follow the on-bead digestion protocol (see below) and submit the digest to your core facility for effective mass spectrometer analysis of (co-) precipitated proteins.
Life science laboratories apply green fluorescent proteins (GFP) to study protein localization, interaction and dynamics in fluorescence microscopy. Immunoprecipitation (IP), mass spectrometry (MS), co-immunoprecipitation (Co-IP) and/or affinity purification investigate more aspects including posttranslational modifications (PTMs), DNA binding, and protein-protein interaction. Here, we compare two different antibody systems for immunoprecipitation of GFP-fusion proteins: GFP-Trap and anti-GFP IgG antibody
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_M 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.