Influencing factors of SDS-PAGE

1. Properties of charged particles <br>How much net charge, particle size and shape. Generally, the net charge is large, the diameter is small and close to the spherical shape, the swimming speed is fast, and vice versa.

2. Electric field strength (potential gradient)
Refers to the unit length (cm) supporting the potential drop on the object, which plays a very important role in the mobility. Generally, the higher the electric field strength, the faster the charged particles move. According to the electric field strength, the electrophoresis can be divided into low pressure (normal pressure) electrophoresis of 100-500 V, electric field intensity of 2-10 V/cm, and separation time of several hours, several days or longer. High-pressure electrophoresis 500-1000V, electric field strength 20-200V/cm, short electrophoresis time. Sometimes it takes only a few minutes, mainly for amino acids, peptides, and nucleotides. The cooling device is required due to an increase in voltage and an increase in current.

3. PH of the solution  
The pH of the solution determines the degree of dissociation of the charged particles. It also determines how much net charge the substance carries. For ampholytes such as amino acids and proteins, the farther the pH of the solution is from the isoelectric point, the more net charge the particles carry, and the faster the electrophoresis speed. The slower the opposite. In serum: albumin pI 4.0; α2 globulin pI 5.06; β globulin pI 5.1; γ globulin pI 7.1. When electrophoresed in a buffer of pH 8.6, it has a negative charge, and its migration rate is: albumin>α2 globulin>β globulin>gamma globulin. In order to facilitate the separation of the protein mixture, a pH value that makes the difference in charge between the various proteins should be selected.

4. Ionic strength of the solution <br>The ionic strength requirement is minimum while maintaining sufficient buffering capacity. The higher the ionic strength of the solution, the slower the moving velocity of the charged particles, and the faster the reverse. Usually chosen between 0.05 and 0.1 mol/L. The ionic strength of the buffer solution can be calculated by the following formula: I = 0.5 Σ cZ2; I is the ionic strength of the solution; c is the ion concentration; Z is the ion valence. Such as: ionic strength of 0.154 mol / L NaCl solution. I = 0.5 (0.154.12+0.154.12) = 0.154.

5. Electroosmosis <br> Another important factor affecting electrophoresis in the electrophoresis of a support is electroosmosis. Electroosmosis: The relative movement of a solution to a solid support in an electric field. Electroosmosis produces electroosmotic flow of varying degrees and directions depending on the support medium. Therefore, electroosmosis is closely related to electrophoresis speed.