Genomic tagging of proteins
We designed and tested a modular epitope system for genomic tagging of the proteins. Our modular tag contains one of the versions of a fluorescent protein for in vivo localization and a small double affinity tag for immunopurification. We continuously optimize the performance of both modules for characterization of the dynamics of protein localizations and interactions.
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Expression of tagged proteins
We express the tagged versions of the proteins we study in Saccharomyces cerevisiae and in HEK293 cells. When we study the yeast proteins, we use a homologous recombination to incorporate our modular tag at the C-terminus of the protein of interest.The tagged proteins are expressed under the control of endogenous promoters.
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Cell-cycle synchronization/cell response
We use a variety of chemicals to force cells to a particular cell-cycle stage. Then we release cells from the action of the chemical to study the dynamics of the cell-cycle progression and/or cell response. We are also interested in developing the new methods for obtaining the synchronous cell cultures.
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Confocal microscopy
We determine the sub-cellular localization of the tagged proteins in the progressing cells at time t0, t1, t2, t3…. etc. We also sample cells at each time point and freeze them in liquid nitrogen. We brake cells under cryogenic conditions and purify the tagged proteins and the protein complexes for the detailed characterization by mass spectrometry.
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Purification of proteins
We purify proteins and protein complexes from cells frozen at time t0, t1, t2, t3…. etc. Information is preserved in frozen cells, but can be lost after thawing. Thus, we develop methods for fast immunopurification (IP) of proteins and protein complexes. Currently we perform a typical IP experiment within 0.5-1 hour, but aim to reduce the IP time to several minutes without compromising the protein yield. More»
Mass spectrometry
We use mass spectrometry to identify proteins and the protein complexes. We also map and profile modification sites on the identified protein components. We also develop and build new mass spectrometric tools for identification of proteins and post-translational modifications. The examples can be found in our published work and the MS Tools section.
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Data analysis
We establish and correlate of the dynamics of sub-cellular localization, the composition of protein complexes and abundances of modification sites. These data give us clues about the mechanisms of dynamic control. Further correlation of the data with the information about the protein structure and protein partners gives us some information about possible processes dynamically controlled by modifications. More»
Conditional changes
We perturb the system and introduce mutations to the studied protein at the sites bearing particular discovered modifications. We repeat the entire round of the experiment with the mutant form of a protein to observe a molecular phenotype. Such molecular phenotypes may give us some clues of how to produce an observable physiological phenotype or a diseased state of the cell. More»
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