Pre-lab Question, Session 2: Which of the following is a consequence of adding SDS to sample buffer and the SDS gel, adding beta-mercaptoethanol to the sample buffer, and boiling the sample before running our SDS-PAGE gel in lab?Group of answer choicesProteins can be separated based on sizeAll of these answers are correctProteins can be separated based on their 3D structureProteins can be separated based on their net charge
Question
Pre-lab Question, Session 2: Which of the following is a consequence of adding SDS to sample buffer and the SDS gel, adding beta-mercaptoethanol to the sample buffer, and boiling the sample before running our SDS-PAGE gel in lab?Group of answer choicesProteins can be separated based on sizeAll of these answers are correctProteins can be separated based on their 3D structureProteins can be separated based on their net charge
Solution
All of these answers are correct.
Here's why:
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Proteins can be separated based on size: SDS (Sodium Dodecyl Sulfate) is a detergent that denatures secondary and non–disulfide-linked tertiary structures, and it also applies a negative charge to each protein in proportion to its mass. This allows proteins to be separated based on their size during SDS-PAGE.
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Proteins can be separated based on their 3D structure: Beta-mercaptoethanol is a reducing agent that is often added to protein samples to disrupt disulfide bonds, thus reducing the protein to its primary structure and eliminating the 3D conformation.
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Proteins can be separated based on their net charge: The SDS denatures the proteins and gives them a uniform negative charge. This means that the separation of proteins in the gel is not based on their original charge, but on their size.
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Boiling the sample ensures that the proteins are fully denatured and that the SDS and beta-mercaptoethanol have fully interacted with the proteins.
So, all the steps mentioned are necessary for the successful running of an SDS-PAGE gel in the lab.
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