Protein+Purification

By Sam V and Mike G
 * = [[image:http://tsienlab.ucsd.edu/HTML/Images/IMAGE%20-%20Rendered%20GFP%20-%20640.jpg width="133" height="133"]] ||= **How difficult is it to purify the GFP protein? ** ||= Why make it as pure as possible? ||= What applications could benefit from a mass production method for GFP? ||

**Driving Question:**

**How can PGLO protein be tagged and extracted MOST EFFICIENTLY for mass production? **

Describe your Topic below:

GFP, or Green Fluorescent Protein, is one of the most innovative proteins within the chemical, medical, and biological fields. It's unique properties allow it to act as a tracer, and indicator, and a tool. Since scientists isolate the PGLO gene coding for GFP, the protein has exploded into uses in genetic disease studies in animals, chemical tracking in physiology and pharmacology, and even [|**surgery**]! For mass production, we need to use protein synthesis.

We can grow proteins in bacteria via genetic engineering (insulin, HGH, etc). We have methods of extracting these proteins: electrophoresis, chromatography, and dialysis. We want to find the most efficient process in extracting GFP, a stand-alone protein with no other metabolic accessories.

Brainstorm what you know and what you would like to know:

> Some are analytical in nature, some are best for mass production.
 * = ** What we (think) we know ** ||=  ||= What we would like to know / understand ||
 * = * We have looked into different methods of protein purification.

> bioluminescent //Aequorea victoria// jellyfish, and that it glows green > when exposed to UV light.
 * We know that GFP is a barrel-shaped, stand-alone protein found

> which can help us purify the protein using its chemical, > polar, and physical properties. ||=  ||= * We'd like to better understand protein purification > by purifying some GFP > works best, quantitatively measuring which method > yields the highest output
 * We also know some of the protein characteristics of GFP,
 * We'd like to test several methods to see which

> real-world applications of protein purification by > using it in a project. ||
 * We would then like to learn about some of the

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Our Lab Video media type="youtube" key="qvz6fqm6m3k" height="315" width="560"

=**Research Links **=


 * Basic information on Proteins and Protein Function: **
 * [|Protein Basics (choose Protein)] **

**Protein Structure: affects how proteins interact and how we can separate them based on these properties** media type="youtube" key="lijQ3a8yUYQ" height="268" width="350"

[|__http://www.westminster.edu/acad/sim/pdf/SpGLOTransformation.pdf__]
 * Great information about pGLO and the instructions for the pGLO transformation lab: **

[|__http://www.pdn.cam.ac.uk/g2o/__]
 * Website showing on genes to organisms: **

[|__http://en.wikipedia.org/wiki/Protein_purification__]
 * Wikipedia on protein purification: **

**Histidine-tag Protein extraction:** **Another great step by step protein extraction video:**

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= = = __//**Taking the Project outside the Classroom: A Visit to NMS Labs**//__ =
 * On Wednesday, April 4th, Sam took a tour of NMS Labs in Willow Grove, PA to further understand protein purification.**
 * While we can explain what he experienced there, we cannot post photos of the visit by company policy.**

==**__NMS Labs:__ A chemical and biological analysis laboratory operating in Willow Grove, PA and another location. The lab runs tests on drug samples, crime scene evidence (blood, bodily fluid, etc), and drug diversion (tampering). The scientists there are known for using cutting-edge methods in all their work, and have been involved in cases such as the drug overdose of Anna Nicole Smith. For more information, visit [|nmslabs.com]**==

===**__Chemistry:__ The scientists uses methods like color testing, liquid chromatography, and [|Gas Chromatography/Mass Spectrometry] to analyze drug samples and verify them against a database.**===

**__Biology:__ The scientists analyze serology (body fluid) samples using microscopy, Electroluminescent microscopy (ID both somatic, gametic cells, and other tissues using different light frequencies), DNA profiling, Polymerase Chain Reaction (even real-time PCR!). They also analyze proteins of interest using Protein Purification, which is why I visited the lab.**

__**Protein Purification (Analytical)**__
I met with **Dr. Christian Westring, Ph.D.**, in the biology department at NMS. Dr. Westring taught me about protein analysis through purification and isolation.

Protein Purification, as we learned, is when you isolate a protein product from a larger sample. Once the proteins have been separated from the cell membrane and other macromolecules, we can analyze them. In analysis, we can use traditional methods like SDS gel electrophoresis/Western Blot or more efficient methods like 2D (2-stage) High Performance Liquid Chromatography (HPLC).


 * SDS gel Electrophoresis ** works just like DNA electrophoresis; you separate proteins based on size. First, add SDS (a detergent) to denature the proteins into their primary structure chains. Then, run the proteins through a charged gel via electrophoresis and ID the proteins based on banding patterns.



**Western Blot** can then be used to identify a single protein from the SDS gel. The proteins are transferred to a sheet containing the specific antibody that binds to the protein of interest. After it binds, we clear the sheet of all other proteins by washing them out, and only the target protein remains.



** 2D HPLC (High Performance Liquid Chromatography) ** is a more efficient method to identify proteins. Essentially, it works by exposing the proteins to increasingly powerful conditions, and separating them based on their own properties (proteomics). The extracted proteins are run through a capillary tube at the same time. We can then use two factors like pH and hydrophobic reactions to split up the proteins. First, we run the sample through one of the conditions, i.e. pH. As time goes on, we add more cations, and this will cause different proteins to run at different rates through the capillary tube. Next, the samples can run through a second condition, i.e. a solvent affinity based on hydrophobia where, again, different proteins pass through at different rates. Finally, when they reach the end of the tube, the machine can detect their presence. Based on **when** they stop, we can ID the protein by Mass Spectrometry (comparing the results to a database).



**Thanks Dr. Westring and Mr. Diamond, and the rest of the supportive staff at NMS Labs!!! :)**

**FINALLY...OUR OWN PROTEIN PURIFICATION!!!**
**Check the Prezi for pictures and videos of our own experiment! Thanks for your time!**