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Chromatographic methods are widely used for studying different objects. Variuos chromatographic equipment with range of detectors, including ms-detectors used in dept. VOC (volatile organic compounds) are studied under GC (gas chromatogpaphy) methods, using HPLC (hugh perfomance liquid chromatography) vitamins, carbohydrates, phenolic compounds, peptides and proteins are examined.
In addtition to chromatographic methods CE (capillary electrophoresis) system also used in the case of extra-small quantity of samples. By CE can be analysed both organic and inorganic compounds (cation/anion).
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Matrix-assisted laser desorption/ionization time-of-flight/time-of-flight tandem mass spectrometer (MALDI-TOF/TOF) Bruker Daltonics Ultraflextreme
The Ultraflex mass spectrometer is designed for automated measurements of mass spectra of substances. The Ultraflex mass spectrometer is used in different cases: physico-chemical studies of substances and materials in biochemistry, biotechnology, physical chemistry, chemistry of synthetic polymers, pharmaceutics and medicine.The device is a multi-purpose automated measuring system consisting of ion source, vacuum chamber, mass analyzer and a personal computer. The mass spectrometer is implemented tandem mode, which is used in more than one stage of mass selection ((MS)n-mode). Ionization is performed by laser radiation interacting with a sample located in the plane of the matrix target (Matrix Assisted Laser Desorbtion/Ionization – MALDI method). Programmable scanning option provides high performance analysis (up to 384 per download). Detection of ions is performed in the time-of-flight analyzer in linear mode and reflection mode. The device is equipped with a LIFT function that allows you to pre-slicing ions. The mass spectrometer Ultraflex allows to determine the mass of product in the range of 500Da to 120kDa, to identify proteins by peptide fingerprint using the Mascot service and pretreatment of the spectra with FlexAnalysis software package. It is also possible to identify amino acid substitutions and protein modifications using the tandem mode of the device. The specialization of our department is the identification of proteins by peptide fingerprint. We also conduct extensive studies of the human proteome, MTB and other model objects.
What information can be obtained from spectrophotomer ?
Electron-vibrational spectrum of molecules in the UV and visible range of the spectrum represents the dependence of the absorption coefficient of monochromatic light of the wave length (frequency). The spectrophotometer measured the absorption of light by molecules is the sum of the net absorption and light scattering.
1. Determination of the concentration.
The most widely used measure of absorption are the absorption coefficients (ε) that is determined from the relation
ε = A/(С∙ l) , where
A — opticaldensity of the sample (measuredbyspectrophotometer),
l — opticalpathlength (cuvettethickness, polymerfilm, etc.),
C — molarconcentration of the sample, orpercentcontent of a substance in solution, polymertape, polymerbloc, etc.
Percentage (by weight) of the absorption coefficient isused in cases when the unknown molar concentration (e.g., proteinswithunknownprimarysequence). When determining the concentration by absorption spectrum it must be remembered that the absorption spectrum have a fundamental dependence on temp, the dielectric properties of the solvent, light scattering. The main sources of measurement error are: a) offset and the curvature of the base line result to the replacement of the cuvette with the solvent in the cuvette with the test solution; b) in accuracy in the determination of the cuvette thickness; c) the shape distortion of the spectrum due to the scattering of the sample.
2. Control of the purity of the specimen.
3. Analysis of multicomponent systems with highly overlap ping bands.
Is the differentiation of the spectrum (the spectrum of the second derivative of the line almost twice narrow), decomposition of spectrum intogaussiancomponents, etc.
4. Dimension spectra of linear dichroism (in oriented samples)
5. Dimension of differential spectrum (oxidized minus reduced, light minus dark, perturbations of temperature, the influence of smalladditives, etc.)
6. Dimension of the kinetics of reactions with detection at a given wavelength
7. Dimension of Rayleigh scattering ( where there is no absorption )
What information can be obtained from a spectro fluorimeter?
Fluorescence is spontaneous emission from the lower singl-etexcited level (S1 →S0 ). The rate constant of this process (kf) is determined from the area under the curve in the spectrum of the absorption band S1 →S0 and is a very stable parameter of the molecule. Competing with the emission without radiative deactivation processes of the singl-etexcited (vibrational and thermal decontamination, transition to triplet state, intramolecular proton transfer, etc.) on the contrary, can depend heavily on properties of the environment (pH, redox potential, viscosity, Association with ot her molecules, without radiative energy transfer, etc.). This feature makes the measurement of fluorescence spectrum is much more flexible and use fulrese arch to olthan the absorption spectrum. The limitation is the tfluoresce predominantly those moleculesthat have a system of conjugated double bonds in hard cycles.
Experimental measurement possibility.
1. The quantumyield of fluorescence is the ratio of the speed of the emissionto the absorption rate of photons. Is measured by comparing the radiation intensity of the test sample with the standard, compares the areaunder the spectralcurves.
2. The fluorescence spectrum of (SF) — distribution of radiation intensity at different wave lengths. Spectr are measured for detection of the fluorescent molecules and determine their concentration. To determine the concentration of the fluorophore requires a comparison with a solution of known concentration.
3. Analys is of multicomponent systems. Fluorescence spectrum on depend on the field of excitation. There fore, selective fluorescence excitation with monochromatic light in different regions of absorption allow stodetect the minimum concentration of fluorophores in complexmixtures.
4. Determining a distance between the fluorescent molecules in the formation of complexesor the presence of fluorophores in the polymerchainmay in effect without radiative energy transfer in the presence of spectral distingueished floorforum (range 10-50).
5. Measurement of polarization of fluorescence allows to evaluate the viscosity of the system (membranes, liposomes, etc.) as in true solutions depolarized fluorescence.
6. Measurement of pH, redox potential, viscosity, etc. is carriedout with the use of specialized probes.
7. Measurement of the kinetics of reactions by changing the concentration or change in the quantumyield of the fluorophore is possible with concentrations by 2-3 orders of magnitude lower than with detectionby absorption.
What information can be obtained from spectroscopy circular dichroism (CD)?
Signal CD — arises from the fact that optically active molecules are characterized by different extinction coefficients for monochromatic light with left — and right-El (right) — circular polarization er. In the result, the difference in optical density:
ΔA =Al—Ar= εlcd —εrcd = (εl—εr)cd = Δε cd
where C – concentration, d the optical path.
The value ΔA is usually very small (Δε 2-4 orders of magnitude less than ε) and the sensitivity of modern spectrophotometers CD reaches 10-5 optical density units. Such sensitivity is achieved by the fact that high-frequency modulator alternately converts a monochromatic plane polarized ray of the light left and right circular polarization. There is a very small intensity modulated measuring light and, accordingly, the current from the photodetector. The electronics filters out and amplifies the modulated component.
On your form, range CD, and coincides with the absorption spectrum (for an isolated electronic transition). However, it can be both positive and negative.
The amplitude of the signal CD does not correlate with the amplitude of the signal in the spectrum of absorption.
1. Definition of isomerism of molecules.
2. Quantitative determination of the composition of the mixture of isomers.
3. The configuration definition aggregates (dimers, trimers, etc.) when the exciton splitting of the electronic levels.
4. Determination of the main secondary structure elements of proteins and peptides. Widely used to monitor the folding of recombinant proteins.
5. Determination of thermostability of proteins and peptides by melting curves (5 to 90).
6. Measurement of the kinetics of the reactions of optically active molecules
7. Structure of nucleic acids. Control of education quadruplexes the aptamers.
Gold nanoparticles conjugated with proteins: Preparation and fields of application
Conjugates between gold nanoparticles (AuNPs) and proteins are used as detecting tools in different formats of immunoassay including immunohistochemistry, Western blot, dot-immunoassay and immunochromatography, and more widely – as reactants for visual detection of biospecific interactions.
Immunohistochemistry is a method to identify proteins in the course of tissues studying that is based on specific antigen-antibody reactions.
Immunoblotting technique includes separation of proteins by electrophoresis, their transfer onto solid carrier and the following detection using labeled antibodies.
Dot-immunoassay is based on the use of porous membrane carrier with immobilized antibodies for cross-flow of tested sample and labeled immunoreagents leading to the formation of detectable immune complexes.
Immunochromatographic assay is based on the lateral-flow movement of some eluent along porous membrane carrier, which leads to the formation of specific immune complexes at different zones of the membrane. AuNPs conjugated with one of immunoreagents (usually with antibodies) are distributed along the membrane, and their presence in certain zones after the assay provides information about the presence or absence of target analyte in the tested sample.
AuNPs with diameter in the range from 5 to 50 nm are commonly used as colored markers. To synthesize the AuNPs, citrate reduction of HAuCl4 is typically applied. The reduction protocol is based on the preparation of boiling aqueous solution of HAuCl4 and the following addition of sodium citrate solution in an amount that depends from the desired particle size.
Antibodies are immobilized on the surface of the obtained AuNPs by adsorption; the AuNP–antibody conjugates are synthesized by this way. The optimum concentration of a protein preparation for its conjugation with AuNPs is set based on the flocculation curve reflecting the aggregation of the conjugates under high ionic strength. To plot this curve, absorbance of aggregating AuNPs is measured at 580 nm using microplate photometer Multiskan EX.
Separation of the formed AuNP–antibody conjugates from non-interacted molecules is carried out using high-speed desktop centrifuge with cooling Allegra 64R, equipped with three rotors.
Description of rotors
Rotor Max RPM
Max RCF (x g)
Max capacity (mL) F1202 30,000 64,400 28,500 58,120 12 х 1.5 F1010 26,000 57,440 22,500 43,020 10 х 10 F0650 21,000 41,420 18,500 32,140 6 х 50
Quality of the AuNP–antibody conjugates influences significantly on the quality and stability of the immunoassay system as a whole. To choose the most effective preparation, such parameters as size of AuNPs, the antibody : AuNP ratio and immobilization mode are varied. The rows of preparations obtained by this variation are tested on their functional activity, i.e. the degree of stored ability to receptor-ligand (antibody-antigen, enzyme-inhibitor, etc.) interactions for immobilized protein. Enzyme immunoassay with the use of microplate photometer Multiskan EX (wavelength 450 nm) and immunochromatographic assay are applied for the given testing.
Fermenters are designed for high-density cultivation of microorganisms under controlled conditions (pressure, temperature, nutrient concentration, pH, etc.) in a closed vessel. The recombinant proteins and other biologically active substances can be synthesized in the exponential growth phase (nucleotides, enzymes, vitamins – primary metabolites) as well as in stationary growth phase (antibiotics, colorants, etc. – so called secondary metabolites). In our center both – batch and feed-batch methods of cultivation are widely used.
- Batch culture
Batch fermentation refers to a partially closed system in which most of the materials required are loaded onto the fermenter, decontaminated before the process starts and then, removed at the end. The only material added and removed during the course of a batch fermentation is the gas exchange and pH control solutions. In this mode of operation, conditions are continuously changing with time, and the fermenter is an unsteady-state system, although in a well-mixed reactor, conditions are supposed to be uniform throughout the reactor at any instant time.
The principal disadvantage of batch processing is the high proportion of unproductive time (down-time) between batches, comprising the charge and discharge of the fermenter vessel, the cleaning, sterilization and re-start process.
- Fed-batch processes
The concept was then extended to the production of other products, such as some enzymes, antibiotics, growth hormones, microbial cells, vitamins, amino acids and other organic acids. Basically, cells are grown under a batch regime for some time, usually until close to the end of the exponential growth phase. At this point, the reactor is fed with a solution of substrates, without the removal of culture fluid. This feed should be balanced enough to keep the growth of the microorganisms at a desired specific growth rate and reducing simultaneously the production of by-products (that can be growth or product production inhibitory and make the system not as effective).
- production of high cell densities due to extension of working time (particularly important in the production of growth-associated products)
- controlled conditions in the provision of substrates during the fermentation, particularly regarding the concentration of specific substrates as for ex. the carbon source
- control over the production of by-products or catabolite repression effects due to limited provision of substrates solely required for product formation
- the mode of operation can overcome and control deviations in the organism’s growth pattern as found in batch fermentation
- allows the replacement of water loss by evaporation
- alternative mode of operation for fermentations leading with toxic substrates (cells can only metabolize a certain quantity at a time) or low solubility compounds
- increase of antibiotic-marked plasmid stability by providing the correspondent antibiotic during the time span of the fermentation
- no additional special piece of equipment is required as compared with the batch fermentation mode of operation
- it requires previous analysis of the microorganism, its requirements and the understanding of its physiology with the productivity
- it requires a substantial amount of operator skill for the set-up, definition and development of the process
- in a cyclic fed-batch culture, care should be taken in the design of the process to ensure that toxins do not accumulate to inhibitory levels and that nutrients other than those incorporated into the feed medium become limiting, Also, if many cycles are run, the accumulation of non-producing or low-producing variants may result.
- the quantities of the components to control must be above the detection limits of the available measuring equipment
The culture medium and the type of fermentation are usually dependent on the following properties of recombinant proteins:
- Accessory to primary or secondary metabolites
- Toxicity to producing cells
- Stability in producing cells
- Structural and functional properties of protein
Recombinant protein expression.
To produce these biopharmaceuticals and other industrially important heterologous proteins, different prokaryotic and eukaryotic expression systems are used. All expression systems have some advantages as well as some disadvantages that should be considered upon selecting which one to use. No universal host cell for optimal biosynthesis of all recombinant proteins has been found so far. Therefore it is important to provide a variety of host-vector expression systems to screen the most suitable expression conditions or host cell. Selection of the best one requires evaluation of several options-from yield to glycosylation, to proper folding, to economics of scale-up, etc.
The well-studied bacterium Escherichia coli is the most common host for recombinant gene expression. This prokaryotic expression system is simple to handle, cost-effective, and enables production of large amounts of heterologous proteins. However, expression of several genes, especially eukaryotic genes, results in production of aggregated and denaturated proteins, localized in inclusion bodies, and only a small fraction matures into the desired native form. Thus, despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins.
The eukaryotic expression systems (Saccharomyces cerevisiae, Pichia pastoris) possess definite advantage as compared to bacterial systems, as they can perform many of post-translational modifications usually performed in eukaryotes, e.g. correct folding, disulphide bond formation, O and N-linked glycosylation and processing of signal sequences. Folding and disulphide bond formation have been identified in some cases as the ‘rate-limiting’ step in production of foreign proteins by yeasts i.e. the ability of the organism to process, fold and secrete the recombinant products determines the productivity of the expression system. The yeast expression system has been successfully used to produce proteins that are highly disulphide-bonded by the strain with overexpression molecular chaperones (heavy chain binding protein (BiP), protein disulfide isomerase (PDI)) and with other modification.
Laboratory system for the cultivation of microorganisms.
1. Two units of fermenters with 4 fermenters in each (8 fermenters).Fermenters equipped with an impellers, air supply, cooling and heating system, sensors for measuring temperature, pH and dissolved oxygen. The pH and pO2 electrodes are sterilizable. Sensor readings are displayed on the screen of the computer monitor. Management of the process of cultivation of microorganisms is carried out using the computer. It is possible to cultivate microorganisms at constant temperature, pH and pO2 values. It is possible to carry out the cultivation of bacteria, yeast and fungi.
2. 8 fermenters with a working volume of 1.5 liters and 2 fermeneters with working volume of 4-8 (with total volume 10 liters). These fermenters are also equipped with impellers, air supply, cooling and heating system, sensors for measuring temperature, pH and dissolved oxygen. The pH and pO2 electrodes are sterilizable. The controller displays readings from all sensors, it is possible to observe the process dynamics and, if necessary, make adjustments. For the preparation of sterile culture medium the system includes autoclaves. For preparation of the inoculum system equipped by shaking flasks cultivator.