Biochem: Separation and Purification of Proteins

Electrophoresis:

  • charge and size are two big factors that determine how a protein will behave

  • anode (+) is on the other side of the cathode (-)

    • proteins with a negative charge will be more attracted to the anode

    • proteins with a positive charge will be more attracted to the cathode

  • smaller proteins move quicker than larger ones because the polyacrylamide gel makes it more difficult for larger things to move through it at a rapid pace

Native PAGE and SDS-PAGE:

  • PAGE stands for polyacrylamide gel electrophoresis

  • native implies that this method allows proteins to remain in their natural, folded state

    • proteins are separated based on size and charge

      • smaller proteins move more quickly than larger ones

  • SDS stands for sodium dodecyl sulfate, a detergent that binds to proteins and unfolds them into long, linear shapes.    

    • gives all proteins a uniform negative charge, regardless of their original charge.

  • SDS-PAGE proteins only move based on size, not charge

    • the larger the protein, the slower it moves

Isoelectric focusing

  • in isoelectric focusing, you are focusing on the isoelectric point of proteins, which is the pH at which a protein carries no net charge

  • zwitterion: amino acid with a neutral charge

  • the method you use when proteins might be smaller in size, but you need to separate the based on their pI

Chromatography

  • has two phases, a stationary phase and a mobile phase

  • paper chromatography: a sample is applied to a special type of paper (the stationary phase) and then dipped in a liquid solvent (the mobile phase)

  • helps identify the purity of something after an extraction by comparing it to the pure version

  • gives a way to separate the components, and sometimes even identify them based on how far they travel

  • size-exlcusion chromatography: proteins are spearated by size, with larger molecules passing through the column faster because they cannot enter the small pores in the stationary phase

  • ion-exchange chromatography: separates proteins based on charge, with positively or negatively charged proteins sticking to the column and eluting only when we change the conditions

  • affinity chromatography: uses a highly specific interaction between a protein and a suitable binding partner bound to the stationary phase, allowing for highly targeted purification

  • thin layer chromatography (TLC):

    • stationary phase is usually a thin layer of a solid spread on a flat surface like glass or plastic

    • mobile phase is typically a liquid solvent

    • small spot of sample is applied at the base of the TLC plate, which is then placed in a shallow container with just the bottom edge submerged

      • solvent started to rise up by capillary action, carrying the sample with it

  • in column chromatography the stationary phase is packed into a vertical column

    • compounds that have less affinity for the stationary phase move quickly through the column, while those that have higher affinity move more slowly.

  • affinity chromatography: takes advantage of the very specific interactions between molecules, such as the interaction between an enzyme and its substrate

  • gas chromatography is very useful to separate, identify, and quantify compounds that can be vaporized without decomposition

  • mass spec is a useful technique that allows us to figure out the mass and structure of molecules by measuring something called the mass-to-charge ratio