Developing A Protein Purification Protocol

Billie Parker 6-14-02

OverviewIntroduction to ChromatographyExtraction of Protein from Cells

Introduction to Green Fluorescent Protein

ResultsConclusions

Introduction to Chromatography Chromatography is used to purify complex mixtures.

We used a resin in a column. The resin binds to certain proteins. This purifies these proteins away from other proteins that do not bind to the resin.

After the other proteins have passed through, the bound proteins can be released.

There are different resins for different substances you want to purify.

Hydrophobic InteractionThe resin binds to water-hating proteins.

Increased salt concentrations promote binding.

The bound protein can be released by lowering the salt.

We tried three different resins to see which one worked the best.

Column Information The resin is packed into the column already.

The substance to be purified passes through the resin and binds to it.

Most molecules do not bind to the resin.

FPLC System Components

The buffers carry the sample through the system.

There are two pumps to move the buffers.

Sample is injected into the valve. Then the sample passes to the column. The UV light detector measures the amount of protein based on absorbance.

The sample is divided into fractions by the fraction collector.

FPLC System

Extraction of Protein from Cells Getting protein out of cells is the first step in purification.

We centrifuged the bacteria to get them out of the growth medium.

We used two different methods to break open the bacteria. We resuspended the cells in detergent. We resuspended the cells and froze them.

Centrifuge

Extraction by Detergent After lysing the cells in detergent, we centrifuged the cells at 10,000 xg for 10 minutes and kept the supernatant.

We used a particular resin that binds to only detergent to purify the detergent from the supernatant.

We put ammonium phosphate in the extract to increase the amount of salt to promote binding to the resin.

Then, we loaded the extract on the column.

Extraction by Freezing We used TE Buffer to resuspend the cells.

We put the cultures into the freezer at -70o for 20 minutes.

We let the cells thaw. Then we centrifuged at 10,000 xg for 10 minutes and kept the supernatant.

We put ammonium phosphate in the extract to increase the amount of salt to promote binding to the resin.

Then, we loaded the extract on the column.

Introduction to GFP GFP is Green Fluorescent Protein.

GFP is from jellyfish.

We used bacteria that were engineered to make GFP.

GFP glows when you shine UV light on it.

Introduction to GFP GFP is Green Fluorescent Protein.

GFP is from jellyfish.

We used bacteria engineered to make GFP.

GFP glows when you shine UV light on it.

ResultsThe graphs will show the amount of protein indicated by UV absorbance.

The salt concentration starts high to allow the GFP to bind then it decreases to let the GFP come out.

GFP was detected by shining UV light on the collected fractions.

First Column Tested Salt shows

the salt concentration in the buffer.

UV measures total protein.

GFP shows fractions that glow.

Second Column Tested Salt shows the

salt concentration in the buffer.

UV measures total protein.

GFP shows fractions that glow.

Third Column Tested Salt shows

the salt concentration in the buffer.

UV measures total protein.

GFP shows fractions that glow.

ConclusionsThe octyl column did not bind to the GFP.

Both the phenyl and butyl columns bound the GFP and eluted it with low salt.

We were unable to conclude which column worked the best because we cannot tell how much protein is in the GFP fractions.