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Chrombody® plasmids: Good things come to those who wait!

  
  
  

This time last spring we started marketing our Chromobody® plasmids. Our customers realized the impact of the technology and were immediately beginning to work with the Chromobodies®. Now, one year later the first papers are being published demonstrating the broad applicability of this intracellular fluorescent antibody technology: 

The first is a paper in Molecular Cell by the Lindqvist Lab at the Karolinska Institutet in Stockholm, Sweden. They developed a FRET-based system for accurate quantification of fluorescence from single cells. By visualizing endogenous PCNA at replication foci with the Cell Cycle Chromobody® they nicely show that the mitotic entry network is linked to the completion of S phase. It does not depend on protein accumulation through G2 but is activated by mitotic phosphorylations at the end of S phase. The method they present allows analyzing live-cell as well as extracting temporal information from fixed cells based on endogenous marker proteins. (Akopyan, K.; Silva Cascales, H.; Hukasova, E., et al. Assessing kinetics from fixed cells reveals activation of the mitotic entry network at the S/G2 transition. Molecular cell. 2014, 53, 843-853. http://dx.doi.org/10.1016/j.molcel.2014.01.031

CCC Publication 2

(original image from Molecular Cell)

The second paper is from the Hawes group within the School of Life Sciences at Oxford Brookes University where they nicely show the use of the Actin-Chromobody® in plant cells. They transferred the Chromobody in the Gateway® system and transiently expressed it inNicotiana tabacum leaves at different Agrobacterium tumefaciensconcentrations. In live cell studies they analyzed cytoskeleton dynamics (inhibition of polymerization and repolymerization) upon treatment with latrunculin B and compared the Actin Chromobody® with conventional labeling probes such as Lifeact. They concluded that Actin Chromobody® is particularly beneficial to study actin dynamics in plant cells as it does label actin without impairing dynamic movement and polymerisation of the actin filaments. (Rocchetti, A.; Hawes, C.; Kriechbaumer, V. Fluorescent labelling of the actin cytoskeleton in plants using a cameloid antibody. Plant Methods. 2014, 10http://dx.doi.org/10.1186/1746-4811-10-12

Actin Chromobody in plants

(original image from Plant Methods)

Last but not least the review article by Philipp Kaiser summarized the “Recent progress in generating intracellular functional antibody fragments to target and trace cellular components in living cells”. This paper gives a comprehensive overview on the application of intrabodies (e.g. nanobody, sdAb, fluorobody, Fab fragment, scFv, DARPin, monobody or fibronectin FN3) for cellular research. Among the different approaches to visualize endogenous proteins the Chromobody®technology is highlighted. Apart from High-Content Screening, other inventive applications of the Chromobody® technology are pointed out such as interfering with proteins/ protein function (e.g. HIV-1 Gag) or protein localization or induction of degradation. (Kaiser, P.D.; Maier, J.; Traenkle, B., et al. Recent progress in generating intracellular functional antibody fragments to target and trace cellular components in living cells. Biochimica et biophysica acta. 2014http://dx.doi.org/10.1016/j.bbapap.2014.04.019

BBA Publication 2

(original image from Biochimica et Biophysica Acta)

Interested in our Lamin-Chromobody® and our Dnmt1-Chromobody®? Looking for more information on our Actin-Chromobody® and our Cell Cycle Chromobody®? Want a custom Chromobody®against a special target? Just write us an email or leave a comment below!

Learn more about Chromobodies®.

 

Posted by Kourosh on Thu, June 5, 2014

 

New Nano-Trap: Focus on MK2!

  
  
  

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

MK2 VersionFollowing 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

The heterodimer of MK2 with p38-alpha/MAPK14 forms a stable complex: molecules are positioned 'face to face' so that the ATP-binding sites of both kinases are at the heterodimer interface.8, 9 Other important interaction partners of MK2 have been identified, such as AKT110, HSP27/HspB111, 12, HSF113, PHC2 and SHC114.

Besides phosphorylation another important regulatory post-translational modification of MK2 is described: Sumoylation, which inhibits the protein kinase activity.15

Recently the inhibition of MK2/3 has been identified as an emerging strategy to manipulate the inflammatory response as a therapeutic option.16-18 Small-molecule pharmaceutical inhibitors SB-203580 or genistein block the activation of MK2.19, 20

All these data, together with the growing number of publication on the complex network organization (http://gopubmed.org/web/gopubmed/) underline the importance of MK2 in biomedical research.21

To enable researches to answer the many open questions regarding MAPK-activated protein kinase 2 faster and easier we have now developed our highly reliable MK2-Trap. MK2-Trap (just like our widely used GFP-Trap®) is based on our high quality alpaca antibody fragments (VHH, sdAb, nanobody) coupled to agarose beads. It facilitates the biochemical analysis of MK2 and its interacting partners. Specifically pull down human, murine or hamster MK2 from any biological sample (e.g. cell extracts or tissue material) irrespective of the phosphorylation status or other modifications. Efficiently perform subsequent analysis like Western Blot, Mass spectroscopy or enzyme activity measurements.

As our collaboration partner Prof. Dr. Matthias Gaestel (Head of Institute of Physiological Chemistry, Hannover Medical University, Germany) stated: “The MK2-Trap beads are great for pull-down from lysate; nearly full depletion of MK2 from the supernatant and also nice Co-IP of p38! Thus the beads will be well suited to purify sufficient material of endogenous MK2 for biochemical analysis.“

In a next step we will soon also introduce our MK2-Chromobody® for the intracellular live-cell analysis of endogenous MK2. Just write me an email or leave a comment below if you are interested in this exciting new research tool and want to learn more!

 

Free MK2-Trap Sample

 

 

1.         Clifton, A.D.; Young, P.R.; Cohen, P. A comparison of the substrate specificity of MAPKAP kinase-2 and MAPKAP kinase-3 and their activation by cytokines and cellular stress. FEBS Lett. 1996, 392, 209-214.

2.         Kobayashi, M.; Nishita, M.; Mishima, T., et al. MAPKAPK-2-mediated LIM-kinase activation is critical for VEGF-induced actin remodeling and cell migration. The EMBO journal. 2006, 25, 713-726.

3.         Manke, I.A.; Nguyen, A.; Lim, D., et al. MAPKAP kinase-2 is a cell cycle checkpoint kinase that regulates the G2/M transition and S phase progression in response to UV irradiation. Molecular cell. 2005, 17, 37-48.

4.         Kopper, F.; Bierwirth, C.; Schon, M., et al. Damage-induced DNA replication stalling relies on MAPK-activated protein kinase 2 activity. Proceedings of the National Academy of Sciences of the United States of America. 2013, 110, 16856-16861.

5.         Ben-Levy, R.; Leighton, I.A.; Doza, Y.N., et al. Identification of novel phosphorylation sites required for activation of MAPKAP kinase-2. The EMBO journal. 1995, 14, 5920-5930.

6.         Meng, W.; Swenson, L.L.; Fitzgibbon, M.J., et al. Structure of mitogen-activated protein kinase-activated protein (MAPKAP) kinase 2 suggests a bifunctional switch that couples kinase activation with nuclear export. J Biol Chem. 2002, 277, 37401-37405.

7.         Reinhardt, H.C.; Hasskamp, P.; Schmedding, I., et al. DNA damage activates a spatially distinct late cytoplasmic cell-cycle checkpoint network controlled by MK2-mediated RNA stabilization. Molecular cell. 2010, 40, 34-49.

8.         ter Haar, E.; Prabhakar, P.; Liu, X., et al. Crystal structure of the p38 alpha-MAPKAP kinase 2 heterodimer. J Biol Chem. 2007, 282, 9733-9739.

9.         White, A.; Pargellis, C.A.; Studts, J.M., et al. Molecular basis of MAPK-activated protein kinase 2:p38 assembly. Proceedings of the National Academy of Sciences of the United States of America. 2007, 104, 6353-6358.

10.       Rane, M.J.; Coxon, P.Y.; Powell, D.W., et al. p38 Kinase-dependent MAPKAPK-2 activation functions as 3-phosphoinositide-dependent kinase-2 for Akt in human neutrophils. J Biol Chem. 2001, 276, 3517-3523.

11.       Stokoe, D.; Engel, K.; Campbell, D.G., et al. Identification of MAPKAP kinase 2 as a major enzyme responsible for the phosphorylation of the small mammalian heat shock proteins. FEBS Lett. 1992, 313, 307-313.

12.       Lavoie, J.N.; Lambert, H.; Hickey, E., et al. Modulation of cellular thermoresistance and actin filament stability accompanies phosphorylation-induced changes in the oligomeric structure of heat shock protein 27. Molecular and cellular biology. 1995, 15, 505-516.

13.       Wang, X.; Khaleque, M.A.; Zhao, M.J., et al. Phosphorylation of HSF1 by MAPK-activated protein kinase 2 on serine 121, inhibits transcriptional activity and promotes HSP90 binding. J Biol Chem. 2006, 281, 782-791.

14.       Yannoni, Y.M.; Gaestel, M.; Lin, L.L. P66(ShcA) interacts with MAPKAP kinase 2 and regulates its activity. FEBS Lett. 2004, 564, 205-211.

15.       Chang, E.; Heo, K.S.; Woo, C.H., et al. MK2 SUMOylation regulates actin filament remodeling and subsequent migration in endothelial cells by inhibiting MK2 kinase and HSP27 phosphorylation. Blood. 2011, 117, 2527-2537.

16.       Ronkina, N.; Kotlyarov, A.; Gaestel, M. MK2 and MK3--a pair of isoenzymes? Frontiers in bioscience : a journal and virtual library. 2008, 13, 5511-5521.

17.       Gaestel, M. What goes up must come down: molecular basis of MAPKAP kinase 2/3-dependent regulation of the inflammatory response and its inhibition. Biological chemistry. 2013, 394, 1301-1315.

18.       Gaestel, M.; Kotlyarov, A.; Kracht, M. Targeting innate immunity protein kinase signalling in inflammation. Nature reviews. Drug discovery. 2009, 8, 480-499.

19.       Maulik, N.; Yoshida, T.; Zu, Y.L., et al. Ischemic preconditioning triggers tyrosine kinase signaling: a potential role for MAPKAP kinase 2. The American journal of physiology. 1998, 275, H1857-1864.

20.       Liao, Q.C.; Xiao, Z.S.; Qin, Y.F., et al. Genistein stimulates osteoblastic differentiation via p38 MAPK-Cbfa1 pathway in bone marrow culture. Acta pharmacologica Sinica. 2007, 28, 1597-1602.

21.       Gaestel, M. MAPKAP kinases - MKs - two's company, three's a crowd. Nature reviews. Molecular cell biology. 2006, 7, 120-130.

Posted by Kourosh on Tue, May 13, 2014

ChromoTek Introduces the new F2H® Kit for Protein-Protein Interaction Analysis

  
  
  

ChromoTek GmbH (Martinsried, Germany) announces the launch of a new research assay for protein-protein interaction (PPI)analysis in live mammalian cells, the Fluorescence Two-Hybrid (F2H®) Kit. This new product is the result of Chromotek's long experience with applying the F2H technology in PPI compound screening for pharmaceutical and biotech customers. The new kit format overcomes limitations of currently available methods and will fascinate biomedical scientists, who investigate protein-protein interactions (PPIs) by its ease of use.

F2H principle

ChromoTek’s F2H® Kit enables effortless analysis of interactions between any GFP- and RFP- tagged protein pairs in living mammalian cells by conventional fluorescence microscopy. It is a fast, simple and quantitative way to characterize PPIs in intracellular environment, screen for PPI inhibitors and evaluate their activity in real time. Unlike available complementation assays, such as split-YFP, F2H® is a fully reversible assay and therefore better suitable for testing PPI antagonists. In comparison to FRET assays, the ingeniously simple optical read-out of F2H® does not require sophisticated equipment, making it much more affordable and at the same time robust and reliable.

“Our mission is to create new superior tools for better research,” comments Dr. Kourosh Zolghadr, Head of R&D and inventor of the F2H® technology. “F2H® Kit completes our toolbox for protein interaction analysis, now covering both in vitro and in vivo approaches. Over 4000 customers worldwide use our GFP-Trap®, successfully analyzing PPIs biochemically. Today we launch the Kit allowing a straight-forward testing of PPIs in living cells by microscopy. Just like our GFP-Trap®, F2H® takes advantage of simple fusions to fluorescent proteins (GFP and RFP), which most cell biologists generate for their proteins of interest anyways. In a speedy one-step procedure, these chimeric constructs can be used for PPI analysis, both biochemically and now also intracellularly.

GFP-Trap® and F2H® Kit save your time and resources. Within one single week you have your pilot experiments completed, and your PPI of interest is validated both in vitro and in vivo,notes Dr. Larisa Yurlova, senior cell biology scientist at ChromoTek. “It also gives you great flexibility: With this toolbox, you can identify protein domains involved in the interaction, screen for intracellularly active inhibitors, visualize their activity and compare their kinetics, etc. There are numerous applications possible!”

For references and more information please follow the link: www.chromotek.com/products

About ChromoTek

ChromoTek products set new benchmarks in cellular research. Established in 2008 as a spin-off from Munich’s Ludwig Maximilian University, ChromoTek 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 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 compound validation, the F2H® assay for protein-protein interaction analysis in living mammalian cells, as well as a selection of conventional monoclonal antibodies. About 4000 customers from all over the world trust in ChromoTek products. 

Download Press release

Posted by Larisa on Mon, Mar 31, 2014

ChromoTek and NMI TT ally to start a new chapter in HCA

  
  
  

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. 

NMI TT logo

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.

mitosis

Chromobodies® are single domain antibodies genetically fused to fluorescent proteins serving as functional nanoprobes in living cells. The Chromobody® technology allows for the first time to trace endogenous intracellular antigens and to visualise dynamic changes of these targets upon intrinsic or extrinsic stimuli in living cells. In combination with disease-relevant cellular models, Chromobodies® can be used to screen compound libraries for desired or adverse effects on endogenous targets in multiwell formats. Likewise, high content analysis facilitates the identification and validation of drug targets.

Currently available Chromobody® cell lines allow real time monitoring of the full cell cycle, apoptotic events, DNA damage and repair as well as morpholological changes of the cytoskeleton in disease-relevant model systems. Further cellular markers are under development and will be launched later in 2014.

"The combination of ChromoTek’s leading analysis technology and our long-standing know-how of genetically modified primary cells adds considerable value to our customers", states Professor Hugo Hämmerle, CEO of NMI TT GmbH. "We are looking forward to opening a new chapter for Chromobodies® in disease-relevant cellular models", says Dr. Marion Jung, Managing Director of ChromoTek.

Under the agreement, NMI TT will develop novel Chromobody® expressing cell types for distribution and co-marketing with ChromoTek. Biotechnology or pharmaceutical companies will be offered Chromobody® cell lines off the shelf as well as lentiviral transductions of their own cell lines to enhance their target discovery and HCA capabilities. In addition, NMI TT will offer services to develop or perform assays on these cells tailored to customer’s needs.

About NMI TT GmbH

Established in 2003, NMI TT GmbH is a fully-owned affiliate of the Natural and Medical Sciences Institute (NMI), providing research departments of pharmaceutical companies with customised services. NMI TT GmbH now offers the generation of stably recombinant, Chromobody®-expressing cell lines using in-licensed lentiviral technology, thereby hugely broadening the scope of Chromobody® application.

About ChromoTek GmbH

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 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 and GFP-/ RFP-Booster to amplify the fluorescent signal of GFP or RFP fusion proteins. ChromoTek's fluorescence based protein-protein interaction assay called Fluorescence-2-Hybrid (F2H®) is available as screening service for commercial customers and as off the shelf kit for academic research. More than 4,000 customers from all over the world trust in ChromoTek products.

Download Press release

Posted by Katrin on Mon, Mar 24, 2014

Protein-protein interactions: see them, screen then, publish!

  
  
  

JBS april coverYes! Our latest paper reporting on screening for protein-protein interaction inhibitors with F2H is now online: http://www.ncbi.nlm.nih.gov/pubmed/24476585.

This is also my first first-author paper from my postdoctoral studies at ChromoTek! Together with Big Pharma (Janssen Pharmaceutica NV) we worked on this project, blind-testing compounds on p53/Mdm2 and p53/Mdm4 interactions, and this was a very rewarding experience. Even more exciting was that our independent results aligned extremely well! All right, coming from basic science, one is just used to things not always working that smoothly. The greater was my surprise how easy and fast it was to screen protein interactions with microscopy here, just using GFP- and RFP-fusion proteins.

F2H PPI inhibition 

Fluorescence images show bait and prey interaction in the cellular F2H assay. 

Upper row: GFP-bait forms a bright green spot in the nucleus of F2H cells. RFP-prey interacts with the GFP-bait and forms a co-localizing red spot. 
Lower row: Upon incubation with an inhibitor red spot disappears, red signal is disperse => interaction is disrupted

In my former lab, cell-based analysis of protein-protein interactions by microscopy would mean trying your luck with FRET. And FRET always came in a package with 4-hour late night slots at the confocal, freezing in that spaghetti-top (own fault) and hoping that the software does not crash and your settings will not go to Nirvana, because you still need 18 more cells to get that statistics. And pray that no one comes up with an idea to try a “couple of inhibitors” of this interaction! In different concentrations!

Anyways, screening for inhibitors of protein-protein interactions went much more efficient with F2H. It takes only half a second to look at a cell to say if it is an interaction or not. You should still check about 50 cells per condition – which all in all takes a few minutes. Thus, for our paper I screened 20 compounds in 8 different concentrations on p53/Mdm2 and p53/Mdm4 interactions. This would have taken me ages with cell-based FRET. Disclosure: to speed things up even more, we let an automated 96-well microscope image the interactions and run high-content analysis.

F2H-Assay proved useful when testing not only small-molecule inhibitors, but also stapled peptides, and for real-time monitoring of protein-protein interaction dynamics in living cells as well. Our collaboration with Sir D. Lane’s group on these topics resulted in a few more papers, all published in 2013: http://www.ncbi.nlm.nih.gov/pubmed/23214419, http://www.ncbi.nlm.nih.gov/pubmed/23653682, http://www.ncbi.nlm.nih.gov/pubmed/24278380.

Being so happy with our F2H success, I really wonder if there are easier ways to see proteins interacting?

Posted by Larisa Yurlova on Thu, Mar 20, 2014

Meet us at #SfN13

  
  
  

Neulogo resized 600

Visit ChromoTek's Alpaca Booth on the Society for Neuroscience Annual Meeting in San Diego, California from Nov 9-13 and win "Alice" our cute, little fluffy Alpaca!

20131110 103640 resized 600

ChromoTek changes distribution in the US

  
  
  

Benefit from great introductory pricing of our new partner, Bulldog Bio!

For years GFP- and RFP-Trap customers in the US had to pay a premium, as our former partner Allele Biotech did not quite match our intended resale prices. As of October 22nd, Allele is no longer our partner for the US - you can aquire our renowned camelid Nano-Traps now from three sources, for nearly half the price:

1. Bulldog Bio is our new official partner in the US, until end of November you can benefit from special introductory pricing.

2. Antibodies-Online has been our partner for some years now.

3. From ChromoTek directly.

  • To keep using the Nano-Traps you have relied on in recent years and that have been cited in more than 400 publications in high ranking journals, we kindly ask you to switch to one of the above options.

Happy nano-trapping from your ChromoTek team!

ChromoTek goes US! Meet us during our Roadshow.

  
  
  
US Alpaka

From October 14th on we will be travelling all across the US to meet with customers and interested scientists to talk about their projects and how an Alpaca might be able to launch their next discovery...

Are you interested in the Alpaca antibody advantage, free samples and a nice chat about your projects? If you are close to one of the following locations, get in touch with us and meet us!

October 15th: MIT, Cambridge

October 16th: John Hopkins University, College Park

October 17th: University of Maryland, College Park

October 18th: NCI, Bethesda

October 21st: Medical University of South Carolina, Charleston

October 22nd: McArdle Laboratory, Madison

October 24th: Northwestern University, Evanston

October 25th: Purdue University, West Lafayette

October 28th: Indiana University, Bloomington

November 1st: MSKCC, New York City

November 4th: Rockefeller University, NYC

November 7th: Parkinson's Institute, Sunnyvale

November 10th to 13th: Society for Neuroscience Annual Meeting, San Diego

November 14th: Salk Institute, La Jolla

November 15th: University of California, Riverside

We present two posters at #MipTec2013 in Basel

  
  
  
Poster MipTec Chromobodies 13 09 resized 600 Poster MipTec F2H 13 09 resized 600
  • Intracellular Fluorescent Alpaca Antibodies (Chromobodies):
    Non-invasive Biomarkers for High-Content Analysis in Mammalian Cells
  • Development of Comparative, Cell-based, Reversible p53:Mdm2 and p53:Mdm4 High-Content Fluorescent Two-Hybrid (F2H) Assays for Drug Screening

Abs are Fab!

  
  
  

This week's Nature product focus features antibodies - if you are reading this, you are of course well aware of the importance of antibodies in today's research. 

However, all antibodies lack the possibility to detect proteins in living cells, as it is necessary to fix cells in order to make them permeable for the relatively large antibodies.

Chromobodies overcome this drawback, as they can be expressed in your cell line resulting in an intracellular antibody that detects endogenous proteins without interfering with the cell's normal processes.

To learn more about these exciting new intracellular antibodies, please visit the Chromobody pages on our website or the Nature product focus.

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