Experiment+4++-+Purification+of+Glutamate+Oxaloacetate+Transaminase+(GOT)+from+Pig+Heart

=**Experiment 4 - Purification of Glutamate Oxaloacetate Transaminase (GOT) from Pig Heart ** = (Or, how I learned to stop worrying and love the bomb..)...wait... what?

Chapter 7: pages 213-232 ||= Porcine GOT is a 94kD dimer (its subunits are 47kD each) with a pI of ~5.5. Various purification techniques will be used to fully purify this protein to homogeneity from blended up pig heart. In this experiment, Heat and Ammonium Sulfate Fractionation are used to purify the GOT. ||
 * ~ Objectives of Experiment ||~ Reading in //Boyer// ||~ Principle of Experiment ||
 * = To learn techniques of protein purification ||= Chapter 11: pages 329-339;

===**Important Things to Remember from this lab: ** ===
 * GOT IS A DIMER!!!
 * [[image:http://imgey.com/images/3ii040jpj.png caption="GOT (See the two domains?)"]]
 * Each subunit is 47kDa
 * GOT is an important enzyme in cell metabolism. Here is a diagram of where it is mainly used (Ignore "GOT1" and "GOT2," they are the same thing essentially)
 * [[image:http://imgey.com/images/3ii0lig.png]]
 * (Hmm... BIOC 3653 / BIOC 3713 anyone?)
 * //**Notice that in this process, one mole of NADH is consumed (per GOT activity)**//
 * Why did we heat the homogenate (ground up pig heart....mmm..yummy)?
 * At high heats, proteins denature (unfold and become ~linear)
 * Before: [[image:http://imgey.com/images/egg.jpg]] After: [[image:http://imgey.com/images/egg14.jpg]]....
 * GOT is special, it can stay folded into its tertiary/quaternary structure at temperatures that most biological proteins cannot (~72-75 deg. C)
 * This denatures most of the other proteins in solution so that they "cook" and we can centrifuge them out
 * We kept the supernatant and proceeded with Ammonium Sulfate Fractionation
 * Ammonium sulfate fractionation
 * Used to separate proteins based on solubility
 * The concentration of (NH4)2SO4 changes the ionic behavior of the protein, and therefore its solubility.
 * Ammonium sulfate saturation levels are increased stepwise and centrifuged each step in between
 * The precipitates/supernatants are then assayed for activity of the protein of interest.
 * Our saturation scheme was as follows:
 * Saturate to 55% ->Centrifuge-> Keep Precipitate Sample, Saturate the Supernatant to 75%-> Centrifuge, keep precipitate sample and supernatant sample.
 * Assay the samples (55P, 75P, 75S); Remember where 55S went? It turned into 75P + 75S
 * 75P turned out to have the highest activity in assay, and therefore we proceeded with it in experiment 5
 * Assay of the samples
 * It should be noted that in our assay we were not DIRECTLY measuring GOT concentration, rather, we were measuring GOT activity by way of NADH concentration.
 * [NADH] changed as it was being consumed. We measured [[image:http://imgey.com/images/eqn536qiq.png]]
 * For every 1 mol NADH consumed, there must be 1 mol of GOT helping consume it. It holds then that:
 * [[image:http://imgey.com/images/eqn536aqa.png]]
 * We have now determined how much GOT is being used (its activity) per mL of GOT solution
 * This can be used to determine how much of the recovered protein in a sample is actually GOT