Transcript Developing A Protein Purification Protocol

Developing A Protein
Purification Protocol
Billie Parker 6-14-02
Overview
 Introduction
to Chromatography
 Extraction of Protein from Cells
 Introduction to Green Fluorescent
Protein
 Results
 Conclusions
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 Interaction
 The
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.

Results
 The
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.
Conclusions
 The
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.