Principle of SDS-PAGE for Determining Protein Molecular Weight
SDS-PAGE determines protein molecular weight based on the ratio of protein molecular weight to charge, allowing for separation. In this process, protein samples are first treated to linearize them and are given a uniform negative charge by adding SDS (sodium dodecyl sulfate). The function of SDS is to disrupt the three-dimensional structure of proteins and replace the original charge with SDS molecules, providing all proteins with the same charge density. Thus, during electrophoresis, the migration speed of protein molecules is only related to their molecular weight and not their original charge or shape.
Next, the treated protein samples are loaded into a polyacrylamide gel for electrophoresis. During electrophoresis, protein molecules are separated based on their size (molecular weight), with smaller protein molecules moving faster towards the positive pole of the gel due to their higher migration rate. By comparing the migration distance of the samples with that of protein standards of known molecular weight, one can estimate the molecular weight of the proteins in the sample. Therefore, the principle of SDS-PAGE for determining protein molecular weight provides important information about the size and composition of proteins.
Common Questions:
Q1: Why is it necessary to treat proteins with SDS during SDS-PAGE electrophoresis?
A: The role of SDS mainly involves disrupting non-covalent bonds within proteins, allowing them to adopt a linear structure while imparting a negative charge. This ensures that during electrophoresis, the migration speed of proteins is only dependent on their molecular weight, independent of their original charge and shape.
Q2: How can the molecular weight of a protein be accurately determined in the principle of SDS-PAGE?
A: When determining protein molecular weight using SDS-PAGE, protein standards of known molecular weight are typically used as a reference. By comparing the electrophoretic migration distances of samples with these standards, the molecular weight of the proteins in the sample can be obtained. There is generally a linear relationship between the migration distance of a protein and the logarithm of its molecular weight. Thus, through graphical analysis, a more accurate molecular weight estimation can be achieved.
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