Biotechnology Laboratory

Tranum Kaur; Ayah Malouhi; Tasfia Tahiat

6 Biotechnology Laboratory

TOPIC – SDS-PAGE Assay

Key Takeaway: The response generated by Chat-GPT was accurate. Provided reference was validated on the Google Scholar. AI offers step-by-step guidance for novice lab operators, explaining the “how”, “when”, and “why” of procedures. It can also identify overlooked issues, offering real-time feedback to improve techniques and enhance lab efficiency.

Chat-GPT general prompt:

Act as a graduate student in a medical biotechnology program at a Canadian University studying human physiology course, and explain the question with relevant references and citations in APA 7th edition style format.

Question: Explain the principle of SDS-PAGE electrophoresis and describe how it is used to separate proteins based on their molecular weight. Include the roles of SDS, polyacrylamide gel, and the reducing agent in the process.

Chat-GPT response:

SDS-PAGE (Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis) is a widely used technique for separating proteins based on their molecular weight. The principle of SDS-PAGE involves applying an electric field to proteins in a polyacrylamide gel, which causes the proteins to migrate through the gel matrix. The speed of migration is primarily influenced by the size of the protein, allowing for the separation of proteins based on their molecular weight.

Key Components and Their Roles:

Sodium Dodecyl Sulfate (SDS): SDS is an anionic detergent that plays a critical role in denaturing proteins, meaning it disrupts non-covalent bonds, unfolding the proteins into linear chains. It also imparts a uniform negative charge to the proteins by binding along their polypeptide backbone. This ensures that the migration of proteins through the gel is dependent only on their size (molecular weight) and not on their native charge or shape.
Polyacrylamide Gel: The polyacrylamide gel acts as a molecular sieve through which proteins migrate. It consists of two layers: a stacking gel (low percentage of acrylamide) and a resolving gel (higher percentage of acrylamide). The resolving gel is where the actual separation of proteins occurs. Smaller proteins can navigate the pores of the gel more easily, thus moving faster, while larger proteins are impeded by the gel matrix. The concentration of the acrylamide can be adjusted to optimize the resolution of proteins of different molecular weights.
Reducing Agent (e.g., β-mercaptoethanol or dithiothreitol, DTT): Proteins often contain disulfide bonds that stabilize their tertiary and quaternary structures. Reducing agents break these disulfide bonds, further denaturing the protein into its individual subunits. This ensures that the migration of the protein through the gel reflects the molecular weight of its monomers, rather than the intact multi-subunit complexes.

Procedure:

Proteins are first treated with SDS and a reducing agent, and then loaded into wells in the polyacrylamide gel. An electric current is applied, causing the negatively charged proteins to migrate towards the anode. As they move through the gel matrix, proteins are separated based on their size, with smaller proteins moving more quickly than larger ones.

After electrophoresis, the separated proteins are typically visualized using a staining technique (e.g., Coomassie Blue or silver staining). The migration distances of the proteins can be compared to a molecular weight standard to estimate their sizes.

Applications:

SDS-PAGE is essential in many areas of biochemistry and molecular biology, such as protein purification, assessing the purity of protein samples, and determining the molecular weight of proteins (Laemmli, 1970).

References:

Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227(5259), 680-685. https://doi.org/10.1038/227680a0

Last Updated November 20, 2024

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