Lab Three: ION EXCHANGE CHROMATOGRAPHY

MMmmm..Spinach!

Part A: Ammonium Sulfate Precipitation

What's Important in this Lab?

• What are we isolating?
• What is Rubisco?
• Is its overall charge Positive or Negative?
• What are the 3 properties used to isolate Rubisco?
• What property does Ammonium Sulfate Precipitation use?

Rubisco, the most abundant protein in plants.

So why do we use OD280 in this lab??

I hope you learned in the Spec lab that the optimal OD for proteins is 280!! Convenient, eh?

So what's the deal with Ammonium Sulfate Precipitation? What in the world is going on???

Let's imagine a love triangle, starring:

Water as Megan Fox

Ammonium Sulfate as Daniel Craig

Rubisco (and other proteins) as Shia Le Beouf

In the beginning, Megan and Shia are in love, attached at the hip by hydrogen bonds.

But along comes Daniel Craig, so big, so buff, and has a way hotter charge!

Megan (water) comes in contact with Daniel (Amm. Sulf). Shia (Rubisco/proteins) unfortunately needed to hit the gym because Daniel's strong charge pulls Megan to him and sends Shia to precipitation.

This leaves the proteins and Rubisco precipitated out in a pellet, defeated by the James Bond of this reaction, Ammonium Sulfate.

vs (rubisco)

So Ammonium Sulfate uses the property of..... SOLUBILITY!

Then we do dialysis where the pellet and supernatant are placed in DI water and the small particles flow out by diffusion....on to Part B.