ChromoBlog

Analyze protein interactions live in mammalian cells

Posted by Christoph Eckert on May 16, 2017 4:18:31 PM

  • Do you have GFP or RFP protein constructs and/or libraries?
  • Do you want to screen agonists or antagonists?
  • Do you want to save time and start your experiment without method set-up?

Just do it. All you need is the ChromoTek F2H Kit Basic: Transfect the bait and the prey plasmids with the Platform Reagent into the F2H cells. Image and quantify your results already at the next day.

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Topics: F2H assay, PPI

How to prepare protein interaction partners for MS analysis

Posted by Christoph Eckert on Apr 24, 2017 11:02:03 AM

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.

 

Download application note

 

The PreOmics iST kit can also be used with other ChromoTek Nano-Traps.

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Topics: GFP-Trap, Immunoprecipitation, Mass spec

Unique Vimentin probes presented at AACR 2017 annual meeting

Posted by Christoph Eckert on Mar 28, 2017 10:00:00 AM

Vimentin dynamics can be effectively visualized using the ChromoTek Vimentin-Chromobody, e.g. to monitor epithelial-mesenchymal transition (EMT). To learn more about this unique probe attend Julia Maier’s talk “Tracing EMT with fluorescent biosensors (Chromobodies) in living lung cancer cells” on April 3, 4:20 pm in Room 144, Level 1 at AACR 2017 in Washington, DC.

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Topics: Chromobodies, Chromobody

Alpaca Nano-Traps for Immunoprecipitation (IP) & Rodent Monoclonals for Western Blot (WB)

Posted by Christoph Eckert on Mar 20, 2017 3:28:14 PM

You obtain the most effective and cleanest IPs of your protein when using ChromoTek’s Nano-Traps (derived from alpaca single domainantibodies). You will see the best protein detetions in WB when applying our highly sensitive monoclonal antibodies.


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Chromatin Immunoprecipitation (ChIP) with RFP-tagged or mCherry-tagged proteins

Posted by Christoph Eckert on Mar 14, 2017 10:18:00 AM

See the advantages of ChromoTek’s RFP-Trap in ChIP applications as demonstrated by Dr. Caroline Rivers, Laboratory for Integrative Neuroscience & Endocrinology, University of Bristol. In absence of suitable antibodies for an inducible hormone receptor, mCherry has been fused to the N-terminus of the receptor. In order to effectively pull down the mCherry fusion protein DNA complex, ChromoTek's RFP-Trap was used.

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Topics: Nano-Traps, ChIP, mCherry, RFP-Trap

The most frequently cited anti-RFP antibody

Posted by Christoph Eckert on Mar 1, 2017 11:43:00 AM

The ChromoTek rat monoclonal anti-RFP antibody 5F8 has been cited for the use in immunofluorescence (IF) and immunohistochemistry (IHC) experiments by 91 peer reviewed articles. It is thus the anti-RFP antibody referenced most often at the database citeAB. This top rank is the result of its performance and quality.

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Topics: RFP Antibody, Immunofluorescence

Actin Chromobody: Visualization of actin dynamics – NOT alteration

Posted by Christoph Eckert on Feb 20, 2017 1:21:56 PM

Everybody really wants to use a detection probe that does not influence its target. For measurement of actin and actin-related functions there are a number of actin visualization probes and techniques available that researchers can select. Hence, one should only apply such actin visualization probes, which do not alter actin dynamics. How do the actin probes for visualization perform? Which probes do interfere with function and which probes do not? Robert Grosse and his team from the Institute of Pharmacology at the Biochemical-Pharmacological Center (BPC), University of Marburg, Germany took the effort and systematically looked into detection technologies of actin filaments in order to avoid potential pitfalls. In the recently published mini review/poster “Actin Visualization at a Glance” (Melak et al. 2017) they have discussed the pros and cons of current probes to visualize actin filaments, i.e. Phalloidin, LifeAct, Utrophin, F-tractin, SiR-actin, GFP-actin, tagged actin, and Actin Chromobody® (see table 1 and figure 1 below). 

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Topics: Chromobodies, Actin Chromobody, cytoskeleton, Actin, live cell imaging

New limit in protein complex stability: GFP-binding protein:GFP complex

Posted by Klaus Herick on Feb 3, 2017 3:56:34 PM

Although traditional IgG antibodies are often used for immunoprecipitation and protein interaction analysis, GFP-binding protein (GBP, ChromoTek gt-250) and other ChromoTek VHHs (http://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.

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Topics: GFP-Trap, Nano-Trap

Visualize the onset of metastasis with Chromobodies

Posted by Christoph Eckert on Oct 4, 2016 4:00:00 PM

The progression of epithelial cancers into metastatic stages is triggered by a cellular process called “epithelial-mesenchymal transition” (EMT). In response to cytokines, tumor cells shut down the expression of epithelial makers including occludin or E-cadherin and gain mesenchymal markers such as N-cadherin or vimentin. Such cells are losing their cell-cell contacts and apical-basal polarity and become highly migratory and invasive. Considering the importance of this process for cancer progression and formation of metastasis, reliable cellular EMT biomarker and versatile detection systems are necessary to develop novel anti-metastatic therapies.

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Topics: Chromobodies

Update US ordering information

Posted by Christoph Eckert on Jul 27, 2016 5:22:56 PM

Dear valued US customer,

 
We have set the new ordering email usaorders@chromotek.com in order to accelerate our US services.
 
Please use this email address or, alternatively place your order via our online portal www.chromotek.com/webshop to benefit from improved delivery times.
 

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