ChromoTek Myc-Trap® is an effectiveness optimized tool for immunoprecipitation (IP/Co-IP) and affinity purification of Myc-tagged proteins. Depending on whether you use a single Myc-tag, i.e. 1xMyc (EQKLISEEDL) or a double Myc-tag fused to the protein of interest, i.e. 2xMyc (EQKLISEEDLEQKLISEEDL) you can adjust your experiment. Here, we introduce the epitope of Myc-Trap to discuss its performance for afore mentioned applications.
- 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.
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.
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.
You obtain the most effective and cleanest IPs of your protein when using ChromoTek’s Nano-Traps (derived from alpaca single domain antibodies). You will see the best protein detetions in WB when applying our highly sensitive monoclonal antibodies.
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.
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. The anti-RFP antibody 5F8 recognizes common RFP variants including mCherry, mPlum, mRFP, mRFPruby.
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).
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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