Protein Interactions in vitro

G. Thoma, M. B. Streiff, A. G. Katopodis, R. O. Duthaler, N. H. Voelcker, C. Ehrhardt, and C. Masson, Non-covalent polyvalent ligands by self-assembly of small glycodendrimers A novel concept for the inhibition of polyvalent carbohydrate-protein interactions in vitro and in vivo, Chem. Eur. J., 12 (2006) 99-117. 

A direct method for testing RNA-protein interaction in vitro is by affinity-precipitation or -chromatography. Two molecules interact if specific purification of one of them leads to co-purification of the other molecule. This approach is often referred to as the pulldown method . Different experimental designs can be used One possibility is to let the RNA-protein complex form in solution under native conditions, and then purify the complex by binding a high affinity tag, present in either the RNA or the protein to beads. The physical separation may be accomplished by low speed centrifugation (batch approach) or by column chromatography (column... 

Piehler, J. (2005) New methodologies for measuring protein interactions in vivo and in vitro. Curr. Opin. Struct. Biol. 15(1), 4-14. 

At last, nucleolin might play a specific role in telomeric replication and maintenance, as suggested by two types of data. First, it binds telomeric repeat (TTAGGG)n in vitro (Ishikawa et al, 1993 Pollice et al, 2000), with a marked preference for the single-stranded form. Secondly, it interacts in vitro and in vivo with hTERT (Khurts et al, 2004), the protein catalytic component of human telom-erase. This interaction takes place both in the cytoplasm and in the nucleolus, where it could promote the assembly of hTERT with the RNA subunit hTERC. As a conclusion, many data regarding the involvement of nucleolin in DNA replication are indirect and an experimental demonstration through knockdown or knockout studies is still awaited. 

Schroter, H. and Bode, J. (1982) The binding sites for large and small high-mobility-group (HMG) proteins. Studies on HMG-nucleosome interactions in vitro. Eur. J. Biochem. 127, 429 36. 

Figure 8-17 Working model of the protein-protein interactions in focal adhesions determined by in vitro binding experiments and immunolocalization. In addition, several interactions are of relatively low affinity in solution but may be enhanced at the membrane surface. Abbreviations are ECM, extracellular matrix PM, plasma membrane p-Tyr- , unknown phosphoty-rosine-containing protein R/E/M, member of the radixin/ezrin/moesin family VASP, vasodilator-stimulated phosphopro-tein. Diagram is modified from Simon et al.285...

Whether we discuss silk, proteins embedded in membranes, or soluble complexes of cytosolic proteins, we must ask questions about interactions. A first step is to identify interactions720-730 among proteins either in vitro or in living cells.731 Proteomic methods, which include the yeast two-hybrid method (Box 29-F), are widely used for this purpose. It is possible to identify large sets of interacting proteins, to identify disease states, to observe effects of drugs, and to compare metabolism among species. 

A search for other agents to modify the dynamics of the Pr -> Pfr transformation, and in particular to influence differentially the reaction intermediates, focused on cellular constituents which presumably interact in vivo with phytochrome. Ubiquitin, an 8.5-kDa protein claimed to bind covalently in vivo to Pfr [160] has now also been found to interact in vitro with Pr in the absence of any other cellular constituent [161]. The protein... 

Hofmann, I., Mertens, C., Brettel, M., Nimmrich, V., Schnolzer, N., and Herrmann, H. (2000). Interaction of plakophilins with desmoplakin and intermediate filament proteins An in vitro analysis./. Cell Sci. 113, 2471-2483. 

Wildenauer DB, Oehlmann CE. 1982. Interaction of cyclophosphamide metabolites with membrane proteins An in vitro study with rabbit liver microsomes and human red blood cells. Effect of thiols. Biochem Pharmacol 31 3535-3541. 

A/PI4Ka 1 (Figure 3) is probably the major enzyme contributing to the previously reported F-actin associated Ptdlns 4-kinase activity (Tan and Boss, 1992 Xu et al., 1992). While it was also reported that PIP5K activity was associated with the actin-enriched fraction from plants, the specific isoform of the enzyme has not been identified (Tan and Boss, 1992). As more specific antibodies and molecular probes become available, it will be important to monitor lipid protein interaction in vivo and in vitro. 

As is generally known, the conformational stability of proteins depends on intramolecular interactions and on environmental factors (the surrounding solvent different low-and high-molecular weight compounds). Often, the thermostability of isolated proteins measured in vitro is unexpectedly low, suggesting that the in vitro conditions are lacking stabilizing factors present in vivo. 

Chishima, T., Miyagi, Y, Li, L., Tan, Y, Baranov, E., Yang, M., Shimada, H., Moossa, A. R. and Hoffman, R. M. (1997e) Use of histoculture and green fluorescent protein to visualize tumor cell host interaction. In Vitro Cell Dev. Biol. Anim. 33, 745-747. 

Kaipatkin, S., Pearlstein, E., Ambrogio, C. and Coller, B. S. (1988). Role of adhesive proteins in platelet tumor interaction in vitro and metastasis formation in vivo. J. Clin. Invest. 81, 1012-1019. 

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