Protein Purification

Last Wednesday (February 27, 2013), Eun Ji taught me the basic steps for protein purification. Protein purification is a series of processes intended to isolate a single type of protein from a complex mixture. Eun Ji had already extracted the protein solution by breaking down E.coli. The whole process was done in a 4C room in order to prevent the proteins from degrading (so cold!!><) We went through the following steps to purify the protein:

1) Column Washing
We first set up two columns and pour some resin in them. Resin (ampiphiles) is a chemical used to attracted proteins to its hydrophobic region. The original resin contains buffer that may destroy the proteins, so we need to wash the resin by replacing the original buffer with our own buffer. 

Resin precipitated at the bottom because the particles are relatively big and close-knit. Resin lose its function once it is dry, so I help Eun Ji check them and add  more buffer every 20 min.

2) Loading Samples
Once the resin was fully washed, we load our protein mixture in the column. During the process, resin binds with the targeted proteins with its hydrophobic region while other proteins leaked out. One can repeat this step several times to make sure all the targeted proteins are bind to resin.

Resin can recognize the tag (red circles) we added to the targeted protein prior to this.

3) Washing
Keep adding buffer to the solution (at least 5x the volume of resin). This step can further purify the resin-protein mixture.

4) Elution
This step is very important. In order to separate the targeted protein from resin, we would add molecules (in this case maltose) that have stronger affinity that could bind with resin. This step would increase the volume of the solution.

5) Concentration + Column Regeneration
Last, we need to condense the solution by taking out the buffer. The resin can be re-used for several times.

Another important thing is that when we extract the proteins from E. coli., the mixture also contains proteinase that would degrade out protein. Thus, we need to add proteinase inhibitor to our buffer in order to prevent this.

Where does this fit in the big map?

Later Eun Ji would test the enzyme activity and determine what condition would maximize the production. She told me that the product she would get from her proteins (ANS and 3GT) reaction is currently used in food coloring (red) and food preservation. I thought I was interesting  that scientists suspected the molecule for being anti-cancer and anti-aging because of its anti-oxidant property!

Even though the process was quite long, and it was pretty cold in the room, I thought it was quite an experience!