Scientific and Educational Methods

Frequently Asked Questions

Analytical methods and enzymolgy

What is the uniqueness of the method of studying of analytical methods and enzymology that you offer?
The offered course includes the enhancement of knowledge in the given field in several stages. Due to the way the kits are assembled, every successive stage is an addition to the previous stage, and every previous stage on the other hand is the basis of the information of the next stage. The enzymes and other biologically active compounds (BAC) are included in concentrations that are necessary to conduct different lab practices. The end result of these works are dependency graphs of different sizes. These graphs are constructed given the data obtained by the experimenter, and they must be situated within the limits necessary for drawing different conclusions. That, in turn, is achieved by the correct dosages of BAC contained in the kits offered to you.

1) Purpose of the Semester
To teach the students to model and determine the activity of enzymes using the invertase as an example

2) Aside from optimums T (temperature) and pH (acidity) what other information is needed for modeling enzyme activity?
Aside from optimums T (temperature) and pH (acidity) that are preceived as known, it is critical to determine the concectration of substrate for activity.

3) How to determine the concentration of [S] substrate?
Substrate concentration [S] = 10Km, where Km is the Michaelis constant.

4) How to determine the Michaelis Km constant?
Km is determined from the dependence of maximum speed of reaction Vmax and substrate [S] concentration. The speed of the reaction is determined by measuring the concenration of glucose produced over time.

5) How to determine glucose concentration?
Glucose concentration is determined from glucose calibration curve.

6) How to construct glucose calibration curve?
Glucose calibration curve is constructed from dependencies of standard solutions of glucose and their optical densities. Thus the overall activity is calculated. Determining specific activity is especially important.

7) How to calculate specific activity of the enzyme?
For determining special activity, it is necessary to determine protein concentration which is calculated from the protein calibration curve. Relationship of values of overall activity to the protein concentration give the specific activity value.

8) How to construct protein calibration curve?
Protein calibration curve is built from dependencies of standard solutions of protein and their optical densities. From this dependency, the protein concentration is calculated.

Ion-exchange chromatography

What is the uniqueness of the method of studying of ion-exhange chromatography that you offer?
The absorbent (protein) is pre-stained, and when adsorbing, it changes the color of the adsorbent from white to blue. In the process of chromatography during the increase in ionic strength the protein is desorbed off the column by using unique technology. The location of desorbtion is colored red - that is, it is possible to visually observe how and where the protein desorbed, and when and how it is washed off. Besides, we use a pre-stained marker, that is visually observed in the process of separation, therefore eliminating the need for additional equipment for the detection of separating components.

Gel filtartion chromatography

What is the uniqueness of the method of studying of gel filtration chromatography that you offer?
Classically, the gel-filtration is conducted on a column sized 1-1.5m. The carrier of type SEPHADEX is used, which separates proteins in different ranges, although for this separation, it requires time from 10 hours to 3 days. For detection, it is also necessary to have an ultraviolet detector, a self-recorder and a collector. In the kits we offer, a colon sized 27 cm is used, as well as a polymer, designed especially to have the ability to separate molecules within 40 minutes on a column of this size.

Thin layer chromatography

What is the uniqueness of the method of studying of thin layer chromatography that you offer?
In the method we offer, pre-stained markers are used. They can separate on short distances. In the process of chromatography, it is possible to observe how the separation into separate components from the injected solution takes place.