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.
Although traditional IgG antibodies are often used for immunoprecipitation and protein interaction analysis, GFP-binding protein (GBP, 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, GBP 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.
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.
ChromoTek GmbH (Munich, Germany) has announced today the signing of a license agreement granting Bayer Pharma AG (Bayer) the right to use ChromoTek's proprietary Cell Cycle Chromobody® Technology for drug profiling by High Content Analysis. Under the terms of the agreement, Bayer will apply Chromobody® technology for real time monitoring of cell cycle progression in drug discovery. Financial terms of the agreement were not disclosed. This agreement follows a collaboration in which ChromoTek provided custom cell line development.
ChromoTek's Chromobody® Technology is a patented, universal live cell imaging technology for high content analysis. Chromobodies are single domain antibodies that have been genetically fused to fluorescent proteins to serve as functional nanoprobes in living cells. The Chromobody® technology allows for the first time to trace endogenous intracellular antigens and to visualize dynamic changes of these targets within living cells. Currently available Chromobodies trace important cellular processes like cell cycle, apoptosis, DNA damage and repair or allow cytoskeletal analysis. The Cell Cycle Chromobody® cell line licensed by Bayer traces the complete cell cycle in real time in live cells in a non-invasive manner while providing full flexibility to multiplex with other procedures.
Dr. Kourosh Zolghadr, Head of R&D at ChromoTek comments: "We are pleased to offer our customers efficient tools for achieving more detailed and physiologically relevant information on the effects of drug candidates on cell cycle progression."
ChromoTek products set new benchmarks for cellular research. Established in 2008 as a spin-off from Munich’s Ludwig Maximilian University and located in Martinsried, Germany’s leading biotech cluster, ChromoTek develops and markets Chromobody® based live cell assays for drug screening and target validation. Other products include immunological and bioimaging reagents such as the GFP- Trap® for the rapid pull-down of GFP fusion proteins, GFP and RFP-Booster to amplify fluorescence signals of GFP or RFP fusion proteins. ChromoTek’s newly developed fluorescence based protein protein interaction assay called Fluorescent-2-Hybrid (F2H® assay) is available as a development service. More than 2.800 customers from all over the world trust in ChromoTek products.
Posted by Katrin on Mar 18, 2013
ChromoTek GmbH (Munich, Germany) offers nanobody based real time assays to visualize and measure the action of candidate drugs or other substances of interest on important regulatory processes in living cells. Presently, four Chromobody® plasmids are available for analyzing the cell cycle, apoptosis, DNA methylation and cytoskeleton dynamics.