In laboratories, the technique known as SDS-PAGE is frequently utilized for the purpose of separating proteins according to their respective molecular weights.
It is one of those methods that is routinely utilized but not always completely understood by the person doing so. Let’s make an attempt to rectify that situation by discussing the operation of the SDS-PAGE. The more you understand the technique, the better! Read more here https://www.nature.com/articles/s41598-022-25934-4,
Here’s what you need to know:
Establishing a Relationship Between the Protein Migration Rate and Molecular Weight
One of the first things you should know about SDS-PAGE is that it typically works by separating proteins based on their molecular weight. This is achieved by taking use of the varying rates at which various proteins move through a sieving matrix in response to an applied electrical field.
Any charged species’ path through an electric field is determined by three factors: the species’ net charge, the species’ molecular radius, and the field strength. However, the issue with natively folded proteins is that the net charge or the radius of the molecule doesn’t depend on the molecular weight.
A protein’s net charge is determined by its molecular radius, which is set by its tertiary structure, and its amino acid composition, which can be written as the sum of the protein’s negative and positive amino acids.
Proteins of the same molecular weight but different charges and three-dimensional forms would, therefore, flow through an electric field at different rates depending on the strength of the field in its natural state.
Proteins cannot be separated in an electric field according to their molecular weight alone unless their tertiary structure is first disrupted by reducing it to a linear molecule, and then the intrinsic net charge of the protein is disguised. This is when the SDS comes into play. Find out more here.
The Function of SDS
SDS, a detergent present in the SDS-PAGE sample buffer, disrupts the tertiary structure of proteins. The procedure relies on this detergent, some boiling, and a reducing agent. This causes the folded proteins to revert to a linear molecular shape.
In addition, SDS imparts a uniformly negative charge to the protein surface, masking the R-groups’ intrinsic charges. As a result of SDS binding to linear proteins relatively evenly (about 1.4 grams of SDS per gram of protein), the charge on the protein is now roughly proportional to its overall molecular weight.
Having SDS in the gel ensures that the linearized proteins will remain in their charge-hidden state throughout the run.
Most significantly influencing the mobility of an SDS-coated protein is its molecular radius. These types of proteins are linear molecules with a width of 18 angstroms and a length that scales with their molecular mass. For this reason, the molecular weight of a protein is directly proportional to its molecular radius, and thus its mobility in an SDS-polyacrylamide gel.
Since these types of proteins have the same charge-to-mass ratio, charge-dependent differential migration will not occur.
When an electrical field is produced, proteins that have been treated with SDS will now flow toward the positive anode with varying velocities based on the molecular weights of the individual proteins. The high-friction environment of a gel matrix will cause the various mobilities to be amplified to a greater extent than they would be otherwise.
Polyacrylamide is utilized as the gel matrix for SDS-PAGE, which is an excellent choice since it is chemically inert and, more importantly, it can be prepared in a variety of concentrations to generate different pore sizes, providing a range of separation conditions that may be tailored to your needs.
The acronym SDS-PAGE refers to sodium dodecyl sulfate–polyacrylamide gel electrophoresis. This technique, as its name suggests, is used in the separation of proteins.
An anionic detergent, such as sodium dodecyl sulfate (SDS), is added to the protein-containing solution prior to analysis. This denatures the proteins by disrupting their secondary and tertiary structures that are not disulfide-linked. Even if their molecular weights were the same, proteins with various folding patterns would migrate at varying rates across the gel matrix in the absence of SDS.
SDS, which linearizes the proteins so that they can be sorted only by their molecular weight eliminates the issue. As most proteins have a mass:charge ratio of around 1.4 g SDS/1.0 g protein. We can assume that the distance of migration through the gel is directly linked to merely the size of the protein. During the electrophoretic run, the protein solution’s migration through the gel can be monitored by adding a tracking dye.