A high background from unspecific binding of proteins is a common problem in immunoprecipitation (IP). In this blog, the origin of background and different optimization strategies are discussed. Furthermore, it is shown how you can achieve a low background when using the ChromoTek GFP-Trap®, a ready to use reagent for IP of a GFP-tagged proteins.
Topics: GFP-Trap, Immunoprecipitation
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:
Immunoprecipitation with GFP-Trap.
I: Input, FT: Flow-Through, B: Bound
Based on the properties of the different matrices (see table), we recommend:
Topics: GFP-Trap, GFP Immunoprecipitation, GFP Nanobody, Dynabeads
Topics: GFP-Trap, GFP Immunoprecipitation, GFP VHH, GFP Nanobody
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.
Topics: GFP-Trap, GFP Immunoprecipitation, GFP VHH, GFP Nanobody, on-bead digestion, GFP mass spec
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
Topics: GFP-Trap, GFP, GFP Immunoprecipitation, Best GFP antibody for IP, Best antibody for immunoprecipitation, GFP VHH, GFP Nanobody
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.
Topics: GFP-Trap, Immunoprecipitation, Mass spec, GFP Immunoprecipitation, GFP MS
Although traditional IgG antibodies are often used for immunoprecipitation and protein interaction analysis, GFP-binding protein (GFP VHH, ChromoTek gt-250) and other ChromoTek VHHs (https://www.chromotek.com/about-us/the-alpaca-antibody-advantage/) easily outcompete those under challenging conditions such as elevated temperatures, varying pH or high denaturant concentrations. In fact, GFP VHH tightly binds GFP-fusion proteins even in 8 M urea at 51 °C, which sets a new limit in protein complex stability, virtually unrivalled by any other capture molecule protein tag pair.
Topics: GFP-Trap, Nano-Trap, GFP, GFP- Binding Protein
Benefit from high binding affinities and extraordinary functional stabilities of Nanobodies
Topics: GFP-Trap, GFP Nanobody, GST Nanobody
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ChromoTek GmbH (Martinsried, Germany) and NMI Technology Transfer GmbH (Reutlingen, Germany) have joined forces to advance ChromoTek ́s proprietary Chromobody® technology in new cellular models. |
| Chromobodies® are new intracellular functional antibodies visualising dynamic changes of target proteins in live cells. With the help of NMI`s outstanding lentiviral transduction technology, Chromobodies® will be introduced into difficult-to-transfect cell lines such as primary cells. The cooperation will establish innovative cell lines available for phenotypic screening, target validation and high content analysis (HCA). The unique combination of Chromobody® nanoprobes and a comprehensive collection of disease-relevant cellular test systems will lift preclinical compound screening and pathway analysis in the early drug discovery process to a new level of performance. |
Topics: Chromobodies, Nano-Traps, Nano-Booster, F2H assay, GFP-Trap, Nano-Trap, high content analysis, HCA, Chromobody
