Protein Gel Electrophoresis
In order to study proteins scientists must first isolate them. While many methods of isolation exist, gel electrophoresis is an MCAT favorite.
Basic Setup
Gel electrophoresis works by running an electrical current through a porous gel-like substance. The gel-like substance acts like one of those crazy bungee obstacles that molecules slowly crawl through. Larger molecules have a harder time progressing forward in the gel and take longer to migrate while smaller molecules move far more quickly. By using standardized comparison molecules scientists can determine the size of a protein or peptide sequence in kilo-daltons (kD). Since amino acids are about 110 daltons on average the size can then be used to determine a rough estimate of how many amino acids or residues are present in a protein.
In both cases, molecules move forward due to their charge and the current which generates an electromotive force that pushes charged molecules along with it. So if a molecule isn’t charged it won’t move anywhere.
While we might be used to thinking of gel electrophoresis as a DNA separation technique it is commonly used for proteins. Specifically, polyacrylamide gel is used with proteins and the whole technique is called polyacrylamide gel electrophoresis or PAGE for short.
PAGE An Electrolytic Cell
PAGE is an elaborate electrolytic cell. Since there is crossover between this topic and electrochemical cells it is worth understanding how to apply our knowledge of electrolytic cells to PAGE as the MCAT can ask us questions that refer to both pieces of content information at once.
As a quick refresher electrochemical cells have a cathode where reduction occurs and an anode where oxidation occurs. In an electrolytic cell the cathode is negatively charged and positively charged molecules are attracted to it while the anode is positively charged and attracts negatively charged molecules. In gel electrophoresis, the protein samples are placed in wells at the top of the gel and migrate to the bottom on the basis of their charge.
Typically the top is the negatively charged cathode and the bottom is the positively charged anode although the order can be reversed depending on protein charge. Once a power source is connected the proteins will begin to migrate at varying speeds due to their mass to charge ratio (m/z). Proteins with large m/z move slowly while proteins with small m/z will move quickly. This makes sense since the smaller the mass the faster the molecule and the smaller the charge the slower the molecule.
Reading a Gel
Native PAGE
Native PAGE runs protein samples through a gel as they are allowing them to separate on the basis of their normal m/z ratio and shape. Here proteins with multiple subunits remain intact and migrate together down the gel leading to the creation of one band.
Furthermore, proteins with spread out conformations will take longer to migrate than more compact ones even if they both have the same m/z ratio. Think about trying to get a large kite through a bungee obstacle versus a kite compressed into a ball.
SDS PAGE
SDS PAGE adds Sodium dodecyl sulfate (SDS) to the gel a chemical compound that interrupts non-covalent interactions. So multimeric proteins will be broken apart into their components unless they are held together by covalently linked disulfide bridges. This can lead to multiple bands if the protein is a heteromer or only one band if it is a homomer. However, the protein will be found further down on the gel than a native gel since it has been broken into smaller pieces.
In addition to disrupting non-covalent interactions, SDS also coats the protein in a uniform negative charge eliminating the effect of charge on protein migration. Therefore SDS PAGE evaluates proteins by size alone.
SDS + Reducing PAGE
Lastly, 2-Mercaptoethanol can be added to SDS PAGE resulting in the breakage of disulfide linkages within a protein. In normal SDS PAGE, only non-covalent interactions were disrupted so some multimeric proteins may have stayed together. However, SDS under reducing conditions changes that and breaks apart any remaining multimeric proteins into their constituent parts.
When comparing a regular SDS PAGE and one under reducing conditions the appearance of additional bands or decrease in protein size indicates the presence of covalent linkages. Again due to the presence of SDS proteins are only being evaluated on the basis of their size.
Isoelectric Focusing
Lastly, proteins can be evaluated using isoelectric focusing which takes a different approach than PAGE. Here proteins are placed in the center of the gel at neutral pH. From there the pH gradually and consistently changes getting more acidic on one side and more basic on the other.
Here the positive terminal of the gel is connected to the acidic side of the gel and the negative terminal is connected to the basic side of the gel. At this point, proteins will begin to migrate on the basis of their charge with positive proteins moving towards the basic end of the gel and negative proteins moving towards the acidic end.
Since positively charged proteins are filled with basic residues they will become deprotonated as they enter the increasingly basic gel until the entire protein is neutral or at its isoelectric point (pI). Once neutral it will stop migrating since it is no longer attracted or repelled by either terminal.
The same will occur with negatively charged proteins however they will migrate towards the acidic end of the gel and become protonated as they reach a low enough pH. Again they will stop at their pI. From this scientists can determine the pI of different proteins and learn more about their charges.