Not only are the EMSAs easier, because you don't have to radioactively label anything, but also the Western blots can be saved for years because there is no chemiluminescence that is decreasing after thirty minutes.
Dr. Andreas von Knethen
Postdoctoral Fellow in the Institute of Biochemistry
Johann Wolfgang Goethe-University in Frankfurt, Germany
Dr. Andreas von Knethen is a Postdoctoral Fellow in the Institute of Biochemistry at Johann Wolfgang Goethe-University in Frankfurt, Germany. His research focuses on immune paralysis, tumor biology, inflammation, and hypoxia.
One of the main focuses in Dr von Knethen's lab is apoptosis of T cells. "In sepsis, you have two phases.The first one is the hyper-inflammatory phase, which is characterized by the release of pro-inflammatory cytokines and other pro-inflammatory markers. The second is the hypoinflammatory phase. This phase is also called immune paralysis because the immune-competent cells are decreased or inactivated and then the body is not able to answer adequately to a second or new infection. Mechanisms leading to immune paralysis during sepsis are associated with T cell apoptosis, and lead to depletion of T cells."
Near-Infrared Fluorescence Technology for Consistent and Dependable Analysis
Dr von Knethen's group uses the LI‑COR Odyssey® to analyze PPAR, a transcription factor responsible for T cell apoptosis during sepsis. Fluorescent electrophoretic mobility shift assays (EMSA) are used to examine binding of PPAR to DNA. The oligonucleotides are fluorescently labeled with IRDye® 700 or IRDye 800, which makes the assays "much more easy. You just run the gel and put it onto the Odyssey, and you can see the DNA binding of the proteins. You don't need to dry the gel and there is no radioactivity."
You can use the red and green colors together to see the total amount of protein and the phosphorylation, to analyze both of them on the same blot.
Quantitative Western blots are also useful. Dr. von Knethen uses the Odyssey to analyze protein phosphorylation with two-color Westerns. "You can use the red and green colors together to see the total amount of protein and the phosphorylation, to analyze both of them on the same blot." For gene knockdown studies, he uses Odyssey Westerns to monitor protein levels. He considers it very important to confirm knockdown at the protein level. "You have to do the Western blot just to prove that the protein is really gone, and not only the mRNA is down-regulated."
He feels that the Odyssey makes things easier in the lab. "Not only are the EMSAs easier, because you don't have to radioactively label anything, but also the Western blots can be saved for years because there is no chemiluminescence that is decreasing after thirty minutes."
We thank Dr von Knethen for his contributions in bio-medical research, and are proud to consider him an Odyssey Expert.
For more information about Dr. von Knethen's research, visit Patho Biochemistry.
Publications resulting from work on the Odyssey
- von Knethen A, Neb H, Morbitzer V, Schmidt MV, Kuhn AM, Kuchler L, Brüne B. (2011) PPARgamma stabilizes HO-1 mRNA in monocytes/ macrophages which affects IFN-beta expression. Free Rad Biol Med, 51:396-405.
- Kuhn AM, Tzieply N, Schmidt MV, von Knethen A, Namgaladze D, Yamamoto M, Brüne B. (2011) Antioxidant signaling via Nrf2 counteracts lipopolysaccharide-mediated inflammatory responses in foam cell macrophages. Free Rad Biol Med, 50:1382-1391.
- Schmidt MV, Paulus P, Kuhn AM, Weigert A, Morbitzer V, Zacharowski K, Kempf V, Brüne B, von Knethen A. (2011) PPARgamma- inducedT-cell apoptosis reduces survival during polymicrobial sepsis. Am J Res Crit Care Med, 184:64-74.
- Barra V, Kuhn AM, von Knethen A, Weigert A, Brüne B. (2011) Apoptotic cell-derived factors induce arginase II expression in murine macrophages by activating ERK5/CREB. Cell Mol Life Sci, 68:1815-1817.
- Weigert A, Cremer S, Schmidt MV, vvon Knethen A, Angioni C, Geisslinger G, Brüne B (2010) Cleavage of sphingosine kinase 2 by caspase-1 provokes its release from apoptotic cells. Blood, 115:3531-3540.
- Jennewein C, von Knethen A, Schmid T, Brüne B (2010) MicroRNA-27b contributes to lipopolysaccharide-mediated peroxisome proliferator-activated receptor gamma (PPARgamma) mRNA de-stabilization. J Biol Chem, 285:11846-11853.
- Namgaladze D, Morbitzer D, von Knethen A, Brüne B (2010) Phospholipase A2-modified low-density lipoprotein activates mac- rophages peroxisome proliferators-activated receptors. Arterioscler Thromb Vasc Biol, 30:313-320.
- von Knethen A, Tzieply N, Jennewein C, Brüne B (2010) Casein kinase-II-dependent phosphorylation of PPARgamma at Ser16 and Ser21 provokes CRM1-mediated shuttling of PPARgamma from the nucleus to the cytosol. J Cell Sci, 163:192-201.
- Namgaladze D, Jennewein C, Preiss S, von Knethen A, Brüne B (2009) Attenuated suppression of the oxidative burst by cells dying in the presence of oxidized low density lipoprotein. J Lipid Res, 50:2173-81.
- Weis N, Weigert A, von Knethen A, Brüne B (2009) Heme oxygenase-1 contributes to an alternative macrophage activation profile induced by apoptotic cell supernatants. Mol Biol Cell, 20:1280-8.
- Jennewein C, Kuhn AM, Schmidt MV, Meilladec-Jullig V, von Knethen A, Gonzalez FJ, Brüne B (2008) Sumoylation of PPAR- gamma by apoptotic cells prevents LPS-induced NCoR removal from kappaB binding sites mediating transrepression of pro- inflammatory cytokines. J Immunol, 181:5646-52.
- von Knethen A, Soller M,Tzieply N, Weigert A, Johann AM, Köhl R, Brüne B (2007) PPARgamma attenuates cytosol to mem- brane translocation of PKCalpha to desensitize monocytes/ macrophages. J Cell Biol, 176:681-694.
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