This video provides a comprehensive review of essential laboratory techniques relevant to the MCAT exam. The presenter walks through a document covering various techniques, explaining their principles, applications, and how they might appear on the MCAT. Key techniques discussed include gel electrophoresis (native PAGE, SDS-PAGE, reducing SDS-PAGE), isoelectric focusing, Western blotting, chromatography (HPLC, GC, ion exchange, affinity, TLC), distillation, PCR, and reducing sugar tests.
Here are some practice questions based on the video content:
Which of the following is true about SDS-PAGE? a) It separates proteins based on their native conformation and charge. b) SDS denatures proteins and coats them with a uniform negative charge, separating them primarily by size. c) It uses a pH gradient to separate proteins based on their isoelectric points. d) Disulfide bonds are broken during the process, separating subunits of a protein.
Answer: b) Explanation: SDS-PAGE uses sodium dodecyl sulfate (SDS) to denature proteins and impart a uniform negative charge-to-mass ratio. This allows for separation primarily based on the size of the polypeptide chains, as larger molecules move slower through the gel matrix. Native PAGE separates based on size, shape, and intrinsic charge, while isoelectric focusing separates based on the isoelectric point (pI). While SDS-PAGE does break non-covalent bonds, it doesn't break disulfide bonds unless a reducing agent is also added (as in reducing SDS-PAGE).
In chromatography, what is the principle behind "like dissolves like"? a) Polar substances dissolve in polar solvents, and non-polar substances dissolve in non-polar solvents. b) Non-polar substances dissolve in polar solvents, and polar substances dissolve in non-polar solvents. c) Only polar substances can be dissolved in any solvent. d) Only non-polar substances can be dissolved in any solvent.
Answer: a) Explanation: The principle of "like dissolves like" is fundamental to understanding separation in chromatography. It means that polar compounds will interact favorably and dissolve in polar stationary or mobile phases, while non-polar compounds will interact favorably and dissolve in non-polar stationary or mobile phases. This differential solubility and interaction is what allows for the separation of components in a mixture.
A protein sample is analyzed using three different gel electrophoresis techniques: Native PAGE, SDS-PAGE, and Reducing SDS-PAGE. The results show the following:
What can be concluded about the protein's structure? a) The protein is a monomer with a molecular weight of 100 kDa. b) The protein is a dimer composed of two identical subunits, each weighing 50 kDa. c) The protein is composed of two subunits, one weighing 40 kDa and the other 60 kDa, linked non-covalently, but the SDS-PAGE did not resolve them. d) The protein is composed of two subunits, one weighing 40 kDa and the other 60 kDa, linked covalently via disulfide bonds.
Answer: d) Explanation: Native PAGE shows the protein in its functional, folded state. SDS-PAGE separates based on mass after denaturation by SDS, breaking non-covalent interactions. If SDS-PAGE shows the same single band as Native PAGE, it suggests the protein is either a single polypeptide chain or its subunits are held together by disulfide bonds that SDS alone doesn't break. Reducing SDS-PAGE uses a reducing agent (like beta-mercaptoethanol) to break disulfide bonds. The observation of separate bands at 40 kDa and 60 kDa in reducing SDS-PAGE, but a single band at 100 kDa in SDS-PAGE (without a reducing agent), indicates that the protein is composed of two subunits (40 kDa and 60 kDa) held together by disulfide bonds.
Which of the following statements accurately describes isoelectric focusing? a) Proteins are separated based on their size and charge, moving towards an electrical field. b) Proteins migrate through a gel with a pH gradient until they reach a point where their net charge is zero (their isoelectric point, pI). c) It utilizes antibodies to specifically detect and quantify target proteins. d) It separates molecules based on their volatility and boiling points.
Answer: b) Explanation: Isoelectric focusing (IEF) separates proteins based on their isoelectric point (pI). A pH gradient is established across a gel or column. Proteins are loaded and migrate under an electric field. Positively charged proteins move towards the cathode, and negatively charged proteins move towards the anode. As they migrate, their protonation state changes with the pH. They stop migrating when they reach the pH at which their net charge is zero (their pI), as they are no longer attracted to either electrode.
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