Deceptively simple multiple choice question from biochemistry midterm: Imagine t

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Deceptively simple multiple choice question from biochemistry midterm:
Imagine that you are studying a protein that controls cholesterol levels. You hypothesize that the protein is destroyed by proteases in response to extracellular signals that lead to increased cholesterol. Which of the following techniques would allow you to most rapidly test whether the protein is destroyed?
A. Affinity chromatography B. Co-immunoprecipitation C. Ion exchange chromatography D. Western blot
All except C seem like they could work, but I picked A. Deceptively simple multiple choice question from biochemistry midterm:
Imagine that you are studying a protein that controls cholesterol levels. You hypothesize that the protein is destroyed by proteases in response to extracellular signals that lead to increased cholesterol. Which of the following techniques would allow you to most rapidly test whether the protein is destroyed?
A. Affinity chromatography B. Co-immunoprecipitation C. Ion exchange chromatography D. Western blot
All except C seem like they could work, but I picked A.
Imagine that you are studying a protein that controls cholesterol levels. You hypothesize that the protein is destroyed by proteases in response to extracellular signals that lead to increased cholesterol. Which of the following techniques would allow you to most rapidly test whether the protein is destroyed?
A. Affinity chromatography B. Co-immunoprecipitation C. Ion exchange chromatography D. Western blot
All except C seem like they could work, but I picked A.

Explanation / Answer

Co-immunoprecipitation identifies protein–protein interactions. Target protein-specific antibodies are used to indirectly bind the proteins, which are already bound to a specific target protein. The protein mixture is incubated with an antibody stabilized to a agarose bead. The protein of interest will bind to the specific antibody. If another protein binds to protein of interest, both proteins will be bound to the antibody and eluted out. The proteins have to be analysed by other techniques such as western blot or mass spectrometry.

Affinity chromatography is based on specific binding interactions between molecules. A ligand is chemically immobilized to a solid support. When a complex mixture is passed through it, molecules that bind the ligand will become bund to the resin. The bound molecule is stripped from the column after washing and then analyzed. It is used for purification of proteins.

Ion exchange chromatography is the separation of molecules based on total charge. The molecules will be passed through resin that can be cationic or anionic. Depending on the charge of the protein, the protein will bind to the appropriate column. After washing, the protein is eluted out.

All the three techniques, will require post analysis methods to identify presence or absence of the protein. These techniques are mostly used for protein purification. Analysis of protein may require subsequent techniques like Western blot or mass spectrophotometry for analysis to negate non specific purification.

Western blot is the most appropriate and fast technique to detect whether the protein is degraded by the protease or not. The proteins sample is separated into its constituent proteins by SDS PAGE. The proteins are transferred to nitrocellulose or PVDF membranes a, probed with the specific antibody. A secondary antibody conjugated to a fluorescent dye or HRP binds the primary antibody. Detection is either by emitted fluorescence or color reaction by enzymatic breakdown of substrate. Absence of a band in the sample as compared to presence of specific band in the control sample, will indicate protein degradation.

Option is D.

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