Protoplasm isoelectric point measurement experiment
Protoplasm's main constituent -- protein for amphoteric compounds, it is in different pH medium, its dissociation is different: when the medium's pH value is more small, the dissociation of this substance tends to (1) type, and vice versa tends to (2) type, if in a certain pH value, the dissociation of this substance reaches equilibrium, the pH of the external medium that is the equipotential point of the substance.
Operation method
Protoplasm isoelectric point measurement experiment
Principle
Protoplasm's main constituent--protein for amphoteric compounds, it is in different pH medium, its dissociation is different: (1) in the acidic medium: (2) in the alkaline medium: when the medium's pH value is more and more small, the dissociation of this substance tends to (1), and vice versa tends to (2), if in a certain pH, the dissociation of this substance reaches the equilibrium, the pH of the external medium is the isoelectric point of the substance. Different plant tissues, because they are not the same protoplasm, so has a different isoelectric point. To measure the isoelectric point of plant cells or tissues, staining can be used. Generally, different dyes with different acidity and alkalinity are used as indicators. Commonly used acidic dyes are aniline red, eosin, etc. The colored part of these dyes is anionic; alkaline dyes are Methylene blue, indigo blue, etc. The molecular formula of Methylene blue is: the colored part of it is cationic. The plant tissue to be tested is put into the mixed solution of aniline red and Methylene blue, when the pH value of the medium is lower than the isoelectric point of the tissue, the positively charged protoplasm will adsorb the colored part of the acidic dyes and show a rose red color; when the pH value of the medium is higher than the isoelectric point of the tissue, the negatively charged protoplasm will adsorb the colored part of the basic dyes and show a bluish color; when the pH value of the medium and the isoelectric point of the tissue are equal, the coloring reaction of protoplasm will be a rose red. When the pH of the medium is equal to the isoelectric point of the tissue, the coloring reaction of the protoplasm is a mixture of red and blue--purple. The isoelectric point of plant tissues is not fixed at a certain value, but has a certain range, often referred to as the range of plant tissues "isoelectric zone". This experiment is based on the above principle to determine the isoelectric point of plant tissue.
Materials and Instruments
Citric acid solution Na2HPO4 solution Aniline red solution Methylene blue solution Move I. Materials and equipment Common Problems The acidic dye in this experiment can also be used eosin, if you use eosin for staining, do not have to prepare a mixture of different pH buffer dye, you can directly take the dye solution (concentration of 0.01 M) equal amount of mixing, staining 20-30 minutes, and then take out the sections were immersed into the corresponding pH buffer soak, half an hour later, you can take out the sections to identify the organization of the The isoelectric region of the tissue can be identified by removing the sections after half an hour. For more product details, please visit Aladdin Scientific website.
Pipette Blade Small Petri dish Small beaker Brush Tweezers Coarse filter paper
Tetrapod yellowing seedlings, broad bean seedlings
5 ml and 1 ml pipettes, razor blades, small petri dishes, small beakers, brushes, tweezers, coarse filter paper
0.1 M citric acid solution, 0.2 M Na2HPO4 solution, 0.1% aniline red solution, 0.1% methylene blue solution
Experimental steps
1. Prepare buffers of different pH according to the following table. Prepare 5 ml of mixed dye, 4 ml of buffer + 0.5 ml of 0.1% aniline red + 0.5 ml of 0.1% methylene blue solution in the following proportions in small petri dishes.
2. Take small sections of embryonic stems of four-season bean seedlings, make freehand sections of 36 slices and fix them in 70% alcohol for 5 minutes, then take out 6 slices and put them into the dye mixture of each buffer solution for 20-30 minutes. The dye was removed and 5 ml of the corresponding pH buffer was immediately added and immersed (to the extent of submerging the tissues) for one hour to observe the change in the color of the slides in each buffer. The preparations taken in the vicinity of the isoelectric zone were observed under light microscope for precise comparison of the isoelectric point of the plant tissues. 