Laboratory of Molecular Biology

Biochemical analysis of proteins

 

The Jagiellonian Center of Innovation offers a broad range of services within the scope of proteomic analysis, based on methods of electrophoresis, chromatography and mass spectrometry.

Electrophoretic analyses

Biochemical analysis of proteins using electrophoretic methods, including SDS-PAGE, 2D, native and Western-Blott electrophoresis.

Chromatographic analyses (WYATT)

The proteomics laboratory of the Jagiellonian Center of Innovation allows optimization and performance of protein separation using SEC-MALS and SEC-MALS-QELS, with Heleos II and DynaPro NanoStar detectors. The use of the detectors enables determination of mass and concentration without the necessity to use standards. Moreover, DLS detector makes it possible to work in cuvette mode and hence provides rapid determinations, using volumes of a few µl, of parameters including:

 

  • absolute and relative molecular mass
  • determination of protein crystallization parameters
  • determination of protein melting temperature
  • measurement of conformation changes in the native environment (by changing the hydration radius Rh parameter)

Mass spectrometry (QTOF)

The Jagiellonian Center of Innovation uses spectroscopic analysis method with Impact II QTOF (Bruker) instrument, with an option of coupling with UHPLC chromatograph. This allows generation of high resolution mass spectra (HRMS) and low fragmentation of the studied molecules. With the use of ProteinScape base and BioTools software from Bruker, the following is possible:

 

  • automation of the process of spectrum search
  • determination of protein structures
  • de novo protein sequencing and sequencing with optimization of results from large matrices

Analysis of intermolecular interactions

 

Based on MST and SPR techniques on Monolith NT.115, Biacore X-100 instruments or by using ELISA testing, the Jagiellonian Center of Innovation offers analyses of intermolecular interactions, such as:

  • protein-protein
  • protein-nucleic acid
  • protein-lipid, protein-sugar
  • protein-low molecular compounds

MST (Monolith NT.115)

The method is based on the phenomenon of microscale thermophoresis (MST) that describes the mobility of molecules in a solution in temperature gradient. This unique technology is based on the use of changes in molecule movement in temperature gradient, hence allowing:

 

  • analysis of protein interactions in solution
  • making the analyses independent of the chemical composition of the solution
  • performing experiments in conditions similar to the native ones

SPR (Biacore X-100)

The phenomenon of Surface Plasmon Resonance makes it possible to determine the strength of real-time intermolecular interactions. The SPR phenomenon is based on oscillations of conducting electrons present in the biosensor under the influence of incident light, which results in absorption of strictly defined amount of energy of the incident light wave, leading to lower energy of the reflected wave. The method enables:

 

  • determination of concentration of the studied analyte
  • determination of affinity of the studied interactions
  • analysis of kinetics and thermodynamics of the studied system
  • determination of association and dissociation constants of the forming complex

ELISA tests

This method is used to detect a protein in a studied material using antibodies. Antibodies are conjugated with an enzyme that after addition of a specific substrate catalyzes a reaction of forming a product that can be assayed spectrophotometrically. The method is commonly used for scientific and diagnostic purposes, including:

 

  • determination of concentration and type of proteins in the studied sample
  • diagnostics of presence of viruses and bacteria
  • diagnostics of auto-immune and cancer diseases

PCR, qPCR, RT-PCR

The Polymerase Chain Reaction allows multiplication of specific DNA fragments in vitro. Real Time-PCR is a quantitative PCR reaction that allows simultaneous multiplication of chosen DNA fragments and monitoring of products formed during subsequent cycles. The Jagiellonian Center of Innovation offers services within the scope of PCR and Real Time PCR optimization using non-specific SYBR Green method or specific method using complementary oligonucleotide probes based on fluorescence resonance energy transfer (FRET) between the donor and quenching molecule. Additionally, we offer RT-PCR (reverse transcription PCR), where RNA is used as a matrix.

 

The PCR technique has many applications in genome research, characterization of gene expression, gene cloning, and clinical diagnostics.

Our clients

Application notes

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