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.
Pull-down of proteins can be difficult, particularly when they are expressed at low levels. Here we present 5 tips, which will considerably improve your IP results.
- Protein concentration matters
The higher the protein concentration, the higher the IP yield. Try to use a concentration as high as possible.It makes a significant difference if the concentration of your protein during IP is 1 nM (low protein expression level), about 50 nM (intracellular endogenous protein expression level, will be diluted by buffer for IP) or 1000 nM (high protein concentration).
MAP kinase-activated protein kinase 2 (MAPKAP-K2, MK2; Gene ID: 9261) is a 400 AA (46kDa) large enzyme that plays a central role mainly in the inflammatory response and cytokines production. It belongs to the serine/threonine-protein kinase family and is also involved in endocytosis, reorganization of the cytoskeleton, cell migration, cell cycle control, chromatin remodeling, DNA damage response and transcriptional regulation.1-4
Following stress, it is phosphorylated (at Thr-222, Ser-272 and Thr-334) and activated by MAP kinase p38-alpha/MAPK14, leading to phosphorylation of substrates.5 Phosphorylation of Thr-334 (located between the kinase domain and the C-terminal regulatory domain) may serve as a switch for MK2 nuclear import and export. Phosphorylated MK2 masks the nuclear localization signal at its C-terminus by binding to p38. It unmasks the nuclear export signal, which is part of the second C-terminal helix packed along the surface of kinase domain C-lobe, and thereby carries p38 to the cytoplasm.6, 7
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.
ChromoTek GmbH (Munich, Germany) and Cell Culture Service GmbH (Hamburg, Germany) have entered into a service, distribution and co-marketing partnership.
Under the agreement, Cell Culture Service will provide Chromobody® cells for direct use in live cell assays. These express fluorescently labeled intracellular antibodies allowing real time monitoring of endogenous intracellular proteins. Biotechnology or pharmaceutical companies will be offered Chromobody® cell lines or transient bulk transfections of own cell lines in assay ready formats to enhance their drug discovery and high throughput screening capabilities.
Chromobodies are single domain antibodies that are 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 in living cells. Currently available cell lines allow the high content analysis of the full cell cycle and real time monitoring of apoptotic events. Small molecule libraries can be screened for desired or adverse effects on a cellular level in multiwell formats. "The combination of dynamic live cell analysis and high content read out closes a gap in drug discovery and I am delighted to facilitate the use of this novel technology for my customers", states Dr. Oliver Wehmeier, Managing Director and President of Cell Culture Service. "We are impressed with CCS’ capabilities to manufacture any type of cell based reagent in any type of cell and assay format for Chromobody® customers " says Dr. Marion Jung, Managing Director of ChromoTek.
Customers who have licensed a cell line from ChromoTek can order these cells from CCS directly. Customers who would like to get access to ChromoTek’s technology for the first time can license the cell line or order assay-ready frozen instant cells via ChromoTek or via CCS.
About CCS Cell Culture Service (now a member of the Evotec group)
For more than eleven years, CCS has been supporting research departments of pharmaceutical and biotechnology companies in their efforts to discover new drug entities by supplying them with custom made functionally validated cell based reagents.
ChromoTek products set new benchmarks for cellular research. The company was established in 2008 as a spin-off from Munich’s Ludwig Maximilian University and is located in Martinsried, Germany’s leading biotech cluster. ChromoTek develops and markets immunological and bioimaging reagents and cellular assays for biomedical research and drug discovery. These include the GFP-Trap® for the rapid pull-down of GFP fusion proteins, GFP and RFP Booster for intensifying the fluorescence signal of GFP or RFP fusion proteins, Chromobody® based live cell assays for screening and validation and a fluorescence based protein-protein interaction assay called Fluorescent-2-Hybrid (F2H®). More than 1.900 customers from all over the world trust in ChromoTek products.
Posted by Katrin on Mar 16, 2012