Contact person: Nataliia Iakovenko
The laboratory has a device for studying molecular aspects of animal ethology. The laboratory is primarily used for analysis of nucleic acids, proteins, and steroid hormones as the "building blocks" of physiological systems that determine the reactions of animals to the environment. The first stage of such an analysis is the preparation of primary samples, for which it is necessary to crush and dissolve animal tissues and then purify and separate organic molecules, which will be analysed qualitatively and quantitatively. Dosing of samples is done by pipetting solutions and suspensions, then adding a measured amount of solvents and buffers. This is done using automatic measuring pipettes in the sterile environment of a fume cupboard.
Crushing of organic tissues with destruction of cell membranes is carried out by ultrasound in a sonicator.
Separation of substances in solutions is carried out using a centrifuge where, under the influence of centrifugal force, heavy particles settle to the bottom and parts of the molecules remain in the solution.
Incubation of solutions above or below room temperature is carried out in a ThermoMixer or with a magnetic mixer with heating. Incubation at room temperature, which requires continuous uniform distribution of the substance in large volumes of solution, is carried out using an orbital shaker.
Before sequencing DNA, it is necessary to amplify it, i.e. perform multiple synthesis using the Taq-polymerase enzyme, which operates at around 72 °C. For this, a PCR cycler is used – a device in which it is possible to programme the desired heating-cooling cycle and repeat it as needed in automatic mode. PCR (Polymerase Chain Reaction) is the abbreviation for this enzymatic reaction, as a result of which we obtain millions of copies of the gene we need. After checking the amplified DNA in the solution with the help of horizontal gel electrophoresis and purification, we send it for sequencing.
Gel electrophoresis is used not only to check PCR results. We use vertical gel electrophoresis to separate proteins according to their molecular weight in an electric current with further processing of the gel using the Western Blot method. Blotting is the transfer of separated proteins in the same apparatus for vertical electrophoresis from a gel to a nitrocellulose or PVDF (polyvinylidene difluoride) membrane. The proteins are then trapped (blocked) on the membrane using an orbital shaker. We make a particular type of protein visible with the help of specific antibodies against this protein (primary antibodies) and others (secondary antibodies). We perform protein detection using membrane scanning on a LI-COR C-Digit scanner.
In the laboratory, using the ELISA (Enzyme-Linked Immuno-Sorbent Assay) or EIA (Enzyme Immunoassay) method, we quantify steroid hormones as indicators of stress in wild and domestic mammals. The principle of this method is an immune-enzymatic reaction to detect the presence of a ligand (the studied substrate) in a liquid sample using antibodies directed against this ligand. The amount of coloured product from such a reaction can be measured spectrophotometrically since the intensity of the coloration correlates to the concentration of the detected substrate. To measure the colour intensity of the solutions, a Life Real photometric microtiter plate reader is used, which can detect light with a wavelength from 340 to 820 nm.
We measure the concentration of substances and cell density in cuvettes, including the determination of the concentration of nucleic acids, proteins, etc., using the WAP Biowave compact spectrophotometer, in very small volumes (from 1 µl). This device not only allows measurements, but also the construction of calibration curves for determining the concentration of unknown solutions.
An inverted fluorescence microscope is also available in the laboratory; this can be used to monitor processes in laboratory cultures of microscopic soil animals, as well as in cultures of various tissues. For example, cell nuclei stained with DAPI to highlight DNA can be observed and photographed using a fluorescence unit directly in cell cultures.