Technique: the concept of ammonium sulfate precipitation

Ammonium sulfate precipitation is a method used to isolate proteins by solubility.
Depending on the sidechain (R group) of the amino acid, the charge of the amino acid can vary.

It can be negatively charged, positively charged, or neutral. 

Neutral amino acids are not soluble, because they do not interact well with water. 
The rule of miscibility: like dissolves like (^_^) 
For example, polar molecules that have the same charges can interact together, while neutral molecules with carbons can interact together.

BUT, if you mix polar and neutral molecules together = DOES NOT DISSOLVE, because they are not alike structurally and chemically.

Water is polar. There is uneven sharing of electrons between the oxygen and the hydrogens. Oxygen is pulling the electrons closer to it, because it is electronegative.Oxygen has a partial negative charge, while the hydrogens have partial positive charges. There is hydrogen bonding between the water molecules, as the oxygen forms weak bonds with the hydrogens of other water molecules.

Rubisco (the protein of interest) is an enzyme that fixes CO2 in the Calvin Benson Cycle in plants for photosynthesis. It is found that Rubisco is very soluble in water.  

Form follows function.  

Rubisco has negative charges from phosphate groups. It also has polar OH groups and a carbonyl (carbon double bond oxygen). Therefore, due to its charges and polarity Rubisco can interact well with water.
UC Davis Chemiwiki. Ribulose 1,5-bisphosphate carboxylase (rubisco). http://chemwiki.ucdavis.edu/Wikitexts/UCD_Chem_124A%3A_Berben/Ribulose_1,5-bisphosphate_carboxylase_%28rubisco%29/Rubisco_1 (accessed date 3/22/2011).


In the technique of ammonium sulfate precipitation, ammonium sulfate is used to precipitate Rubisco out of solution in order to isolate Rubisco by solubility. Ammonium sulfate has two positive ammonium groups and four electronegative oxygens.
chemexper. ammonium sulfate. http://www.chemexper.com/chemicals/supplier/cas/7783-20-2.html (accessed date 3/22/2011)

Compared to Rubisco, which does not have a positively charged group, ammonium sulfate seems more attractive to water. 

Once ammonium sulfate is placed in the solution that has Rubisco, the ammonium sulfate will draw all of the water molecules to it, leaving Rubisco out of solution. Therefore, Rubisco will clump together, and with a little help from the centrifuge, you can visually see where Rubisco might be in a pellet at the bottom of the tube.

Rubisco is too soluble, so the ammonium sulfate precipitation has to be perform in two steps.

1. Use a small amount of ammonium sulfate (30%) to extract unwanted proteins that are not very soluble.
2. Use the supernatant (since Rubisco is very soluble, it should still be in solution) to add a larger amount of ammonium sulfate (50%)  to precipitate out Rubisco in the second pellet.

a cool video on translation

Haha, perusing the internet again, and I found this gem. This video is not about protein isolation, but it is a unique and fun way to explain the synthesis of proteins. It's a bit long, but it's worth it to watch!

According to the description, this video was directed (1971) by Robert Alan Weiss for the Department of Chemistry of Stanford University, and it was narrated by Paul Berg, 1980 Nobel prize for Chemistry.

I take no credit for this video. I am only sharing it.

Why do we isolate proteins?

Proteins perform many functions. There is still a lot of mysteries concerning the mechanisms and pathways of most proteins.

Proteins are isolated:

(1) for characterization (to understand its properties)

(2) to understand its involvement in biological systems (What would happen if the protein is absent?)

(3) to address many health issues that is a result of proteins gone awry

Hahaha, while perusing the internet for an informative picture/video. I stumbled upon this interesting video that is center around the isolation of Rubsico (the original reason why this blog exists).



I do not take credit for this video. I am only sharing.

Proteins - they're everywhere!

One amino acid does not a protein make—let alone a being. ~ Preston Cloud

HELLO and welcome to my blog about protein isolation and the various techniques that are used in biology!

Proteins are pretty amazing, because a majority of biological material are made up of them. 

Proteins are polymers of amino acids. 

many groups of amino acids = a protein

When you join many amino acids together, it makes up an entity called a "protein."

http://upload.wikimedia.org/wikipedia/commons/a/a6/Protein-structure.png

Amino acids have a nitrogen-carbon-carbon backbone. 

A hydrogen and an R group (R stands for any sidechain) bind to the center carbon. 

Due to the variability in the sidechain, the property (i.e. the charge) of the amino acid can change to be either positive, negative, or neutral.

Therefore, the structure and function of the protein can change as a result of the properties of the amino acids.

Proteins are everywhere!

Proteins can function as:

1. enzymes

2. hormones

3. transport proteins (transporters or protein channels in the cell membrane)

4. antibodies

5. structural proteins (collage, elastin, keratin, etc.)

6. motor proteins

7. receptors

8. signalling proteins

*Protein Crystallography. Proteins, Proteins. http://www.proteincrystallography.org/protein/ (accessed date 3/18/2011).