We shared several considerations last week for using cellulose acetate membrane electrophoresis, and we will finish this topic here today for your reference.
Selection of Buffer Concentration
The buffer concentration used in cellulose acetate membrane electrophoresis is generally lower than that used in paper electrophoresis. The commonly used pH 8.6 Barbital buffer is typically chosen within the range of 0.05 mol/L to 0.09 mol/L. When selecting the concentration, a preliminary determination is made. For example, if the length of the membrane strip between the electrodes in the electrophoresis chamber is 8-10cm, a voltage of 25V is required per centimeter of membrane length, and the current intensity should be 0.4-0.5 mA per centimeter of membrane width. If these values are not achieved or exceeded during electrophoresis, the buffer concentration should be increased or diluted.
An excessively low buffer concentration will result in a rapid movement of the bands and an increase in band width. On the other hand, an excessively high buffer concentration will slow down the band migration, making it difficult to distinguish certain separation bands.
It should be noted that in cellulose acetate membrane electrophoresis, a significant portion of the current is conducted through the sample, which generates a considerable amount of heat. Sometimes, the chosen buffer concentration may be considered appropriate. However, under conditions of increased environmental temperature or when using higher voltage, the evaporation of water due to the heat can intensify, resulting in an excessively high buffer concentration and even causing the membrane to dry out.
Sample Volume
In cellulose acetate membrane electrophoresis, the amount of sample volume is determined by various factors, including electrophoresis conditions, properties of the sample itself, staining methods, and detection techniques. As a general principle, the more sensitive the detection method, the smaller the sample volume can be, which is advantageous for separation. If the sample volume is excessive, the electrophoretic separation patterns may not be clear, and staining can also be time-consuming. However, when quantitatively analyzing the separated stained bands using elution colorimetric detection methods, the sample volume should not be too small, as it can result in lower absorbance values for certain components, leading to higher errors in calculating their content. In such cases, the sample volume should be appropriately increased.
Typically, the sample volume added on each centimeter of the sample application line ranges from 0.1 to 5 μL, equivalent to a sample amount of 5 to 1000 μg. For example, in routine serum protein electrophoresis analysis, the sample volume added on each centimeter of the application line generally does not exceed 1 μL, equivalent to 60 to 80 μg of protein. However, when analyzing lipoproteins or glycoproteins using the same electrophoresis method, the sample volume needs to be correspondingly increased.
In conclusion, the most suitable sample volume should be selected based on specific conditions through a series of preliminary experiments.
Selection of Staining Solution
The separated bands in cellulose acetate membrane electrophoresis are typically stained before detection. Different sample components require different staining methods, and the staining methods suitable for cellulose acetate membrane electrophoresis may not be entirely applicable to filter paper.
There are three major principles to choose staining solution for cellulose acetate membrane. Firstly, Water-soluble dyes should be preferred over alcohol-soluble dyes to avoid membrane shrinkage and deformation caused by the latter’s staining solution. After staining, it is important to rinse the membrane with water and minimize the staining duration. Otherwise, the membrane may become curled or shrunken, which would affect subsequent detection.
Secondly, it is preferable to select dyes with strong staining affinity for the sample. In cellulose acetate membrane electrophoresis of serum proteins, amino black 10B is commonly used because of its strong staining affinity for various serum protein components and its stability.
Thirdly, reliable quality dyes should be chosen. Some dyes, despite having the same name, may contain impurities that result in a particularly dark background after staining. This can even blur the originally well-separated bands, making them difficult to distinguish.
Lastly, the choice of staining solution concentration is important. Theoretically, it might seem that a higher staining solution concentration would lead to more thorough staining of sample components and better staining results. However, this is not the case. The binding affinity between the sample components and the dye has a certain limit, which does not increase with the increase in staining solution concentration. On the contrary, an excessively high staining solution concentration not only wastes the dye but also makes it difficult to achieve a clear background. Moreover, when the color intensity reaches a certain maximum value, the dye’s absorbance curve does not follow a linear relationship, especially in quantitative measurements.In cellulose acetate membrane electrophoresis, the staining solution concentration is generally lower than that used in paper electrophoresis.
Details to know about Beijing Liuyi Biotechnology’s cellulose acetate membrane electrophoresis tank and its electrophoresis application, please visit here:
l Experiment for separating serum protein by Cellulose Acetate Membrane
l Cellulose Acetate Membrane Electrophoresis
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Reference: Electrophoresis(Second edition) by Mr. Li
Post time: Jun-06-2023