Transcript Protein purification

Protein Purification Tutorial
Protein purification tutorial
TITLE PAGE
graphic
"Proteins" a word derived from the Greek word, Proteios,
which means "of the first rank" was coined by Jons J.
Berzelius in 1938 to stress the importance of this group of
organic compounds. Proteins have been described as the
servants of life. Most proteins are enzymes but in addition
there are also regulatory and structural proteins.
information in these genomes has dramatically changed research. If you can identify
the gene
within
the
Intro –Why
purify
proteins?
Protein
Purification
Tutorial
genomic DNA sequence, you already know the amino acid sequence of the encoded protein and the
regulatory elements that control the expression of this gene. You don’t, however, know the structural
organization of the protein or how it is regulated. If you do not have a genomic database or cannot identify
the protein coding region within the genome, you may want to purify the protein in order to help you to isolate
the gene.
– 1) If you have not yet isolated the gene that encodes the protein, you may want to purify the protein for
any of the following reasons:
– The purified protein can be used to determine the amino acid sequence. The sequence can then be
used as a probe to help in the isolation of the gene.
– The purified protein can be used to produce antibodies that can be used as a probe to help in the
isolation of the gene.
– The purified protein can be analyzed by mass spectroscopy. The molecular weight can then be used as
a screen of the genomic sequence for the gene.
– 2) If you have already isolated the gene that encodes the protein, you may want to purify the protein for
any of the following reasons:
– Pure proteins are required for structural analysis.(x-ray crystallography and NMR spectroscopy)(Figure
1).
– Pure proteins are required to study enzyme function.
– Pure proteins are needed in order to learn about what other proteins or nucleic acids they interacts with.
(Figure 2)
– Pure proteins are needed for studies of enzyme regulation (are their regulatory subunits, is it regulated
by phosphorylation, is the protein regulated by interaction with other proteins.)
Protein Purification Tutorial
• In this tutorial you will:
–
–
–
–
–
1
2
3
4
5
Objectives
Intro – table of common techniques
Protein Purification Tutorial
Table of common methods of protein purification
•
Property
solubility
Methods
Precipitation with
ammonium sulfate
(salting out)*
Size / shape
Size-exclusion
chromotography
Isoelectricpoint
(charge)
Ion exhange
chromatography
binding to small
molecules
Affinity
chromatography
•
Purification procedures attempt
maintain the protein in native fo
Although some proteins can be
natured, most cannot!
To purify a protein from a mixtu
biochemists exploit the ways th
individual proteins differ from o
another. They differ in:
– Thermal stability*
*Ammonium sulfate precipitation is cheap, easy, and accommodates large sample sizes.
It is commonly one of the first steps in a purification scheme.
*For most protein purifications, all steps
are carried out at ~5°C to slow down
degradative processes.
Protein Purification Tutorial
Intro side bar
Picture of protein gel with lanes
showing sequential purification
steps
Purification Table
Procedure
Fraction vol
(ml)
Total Prot
(mg)
Activity
(units)
Specific activity
Units/mg
Crude cellular
extract
1400
10000
100000
10
Purificati Yield
on factor
1
100%
Size-exclusion
90
400
80000
200
20
Ion exchange
80
100
60,000
600
3
Add row with ammonium sulfate data. Include two
colums at end called Purification factor and Yield.
Note: The type and order of steps are customized for each
protein to be purified. An effective purification step results in
a high yield (minimal loss of enzyme activity) and large
purification factor (large increase in specific activity).
80%
75%
Intro – flow chart. Purification is a multi step
procedure
Protein Purification Tutorial
Purification is a multi-step procedure.
Sample
Separation
technique
Repeat with another
separation
technique until pure
Fractionation
No
Assay total protein
Assay enzyme activity
Set aside
No
Is there activity?
yes
Combine
Fractions
Monitor purity
Pure?
yes
Prepare for analytical technique
Protein Purification Tutorial
•
First steps
– 1. Source. A good source is
cheap and readily available.
Many proteins are enriched in
specific tissues, for example
hemoglobin in blood. For this
reason, these tissues may be
excellent sources for your protein.
– 2. Assay: Most assays are
chemical reactions catalyzed by
specific enzymatic activities.
Proteins that have no activity are
usually assayed using SDS
polyacrylamide gels.
Intro – first steps.
Protein Purification Tutorial
•
First steps – Develop an Assay
1. An assay for an enzyme is a
method for quantifying its activity.
Since the assay is repeated many
times, it is important that it be a
simple procedure. Usually
enzyme activity is monitored as a
change in absorbance which can
be measured using a
spectrophotometer. For example
an assay for ribonuclease
measures the change in
absorbance that accompanies the
breakdown of RNA to
ribonucleotides.
Assay – develop an assay
Protein Purification Tutorial
Intro – Preparing the sample (Crude Extract)
First steps: Preparing the sample – Crude extract.
Protein from cells or tissue
Supernatant with
Soluble protein
Break cells,
tissue, or organ
Microbial cells
or tissue
Blender,
homogenizer,
sonication,
pressure,
psmotic
Pellet with intact cells, organelles, mem
Protein Purification Tutorial
•
Some kind of graphic.
Segway to Chromotography
•
Note: In order to isolate sufficient
quantities of protein, you may
need to start with kilogram
quantities of source tissues.
These amounts can best be
handled using precipitation
methods (e.g. ammonium sulfate
precipitation). Later in the
purification, large columns can be
used to handle gram to milligram
quantities. Amounts handled on
gels are typically in microgram
quantities.
Intro – flow chart. Purification is a multi step
procedure
Protein Purification Tutorial
Purification is a multi-step procedure.
Sample
Separation
technique
Repeat with another
separation
technique until pure
Fractionation
No
Assay total protein
Assay enzyme activity
Set aside
No
Is there activity?
yes
Combine
Fractions
Monitor purity
Pure?
yes
Prepare for analytical technique
Protein Purification Tutorial
Column Chromotography –general case
Over view of the apparatus
–
–
–
–
–
–
Glass column
Reservoir
Solid matrix – beads
Solution of protein.
Effluent.
Other terms?
We think it would be better to have a resevoir
attached to a cap on the top of the column via a
plastic tube, show minimal liquid on top of the
column bed, show a tube coming out of the
bottom of the column that has a clamp of
stopcock. Perhaps a schematic on the left and a
photo of an actual column running in a cold box
over a fraction collector on the right.
Should emphasize that the basic set=up is the
same for all column types, but the
characteristics of the beads vary.
Protein Purification Tutorial
•
Image of apparatus with protein
mixture layered on top
Load sample (4 protein mix)
•
TEXT. The crude extract is placed
on top of the solid matrix. (In this
case we are using a mixture of 4
proteins, indicated by different
colors.)
• (As the animation proceeds)
The proteins move at different rates
through the matrix based on the
properties of the proteins and the
type of column beads.
Note - this should be shown as
a single band (possibly brown or
striped with four colors)
Protein Purification Tutorial
•
Fractionation
Collect fractions.
• Text:
As the column separates the proteins
in the mixture, the “effluent” drips
into a series of fraction tubes that
are moving at a specific rate of
speed. These tubes are called
fractions.
Here we are showing 20 tubes.
Fraction collectors in most labs
have about 75-200 tubes.
Be sure to remove color from
column as it drips into the tubes
below! If the sample is spread over
three tubes, the center tube will be
darker in color.
Protein Purification Tutorial
•
In reality, proteins aren’t colorcoded so we must ask ourselves
these 3 questions:
--3 questions—
1. How do we know which fractions
contain protein?
2. Which of those fractions contain
the desired protein?
3. How do we assess the purity?
???
Question 1 – take A280 Screen 1.
Protein Purification Tutorial
Question 1. How do we know which fractions contain protein?
•
Fraction
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
#
Total protein a can be estimated
by taking the absorbance at 280
nm in a spectrophotometer.
Aromatic amino acids absorb light
at this wavelength causing all
proteins Take
to have
A280 absorbance at
280nm. Many fraction collectors
measure the A280 as the column
is running.
Question 1 – take A280 Screen 2 .
Protein Purification Tutorial
Question 1. How do we know which fractions contain protein?
•
Fraction
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
#
A280
0 0 0 2 5 2 0 0 0 2 5 8 5 2 0 0 2 5 2 0
•
Total protein a can be estimated
by taking the absorbance at 280
nm in a spectrophotometer.
Aromatic amino acids absorb light
at this wavelength causing all
proteins to have absorbance at
280nm. Many fraction collectors
measure the A280 as the column
is running.
The A280values
Plot valuescan be plotted
against the fraction number in is
what is called an elution profile.
Question 1 – take A280 Screen 3 .
Protein Purification Tutorial
Question 1. How do we know which fractions contain protein?
•
•
Fraction
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
#
A280
0 0 0 2 5 2 0 0 0 2 5 8 5 2 0 0 2 5 2 0
•
Peaks
A280
Fraction #
Total protein a can be estimated
by taking the absorbance at 280
nm in a spectrophotometer.
The values can be plotted against
the fraction number in is what is
called an elution profile.
Notice the peaks on the graph.
These indicate where the fractions
are that contain protein.
Question 1 – take A280 Screen 4 .
Protein Purification Tutorial
Question 1. How do we know which fractions contain protein?
•
•
Fraction
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
#
A280
0 0 0 2 5 2 0 0 0 2 5 8 5 2 0 0 2 5 2 0
•
Peaks
A280
Fraction #
Total protein a can be estimated
by taking the absorbance at 280
nm in a spectrophotometer.
The values can be plotted against
the fraction number in is what is
called an elution profile.
Notice the peaks on the graph.
These indicate where the fractions
are that contain protein.
Question 2 – take A280 Screen 1 .
Protein Purification Tutorial
Question 2. Which fractions contained the desired protein?
•
Enzyme activity can be
determined by performing an
enzyme assay on each fraction
that contains protein.
Fraction
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
# Fraction
#
0 0 0 2 5 2 0 0 0 2 5 8 5 2 0 0 2 5 2 0
A280
Enz. Assay.
A280
Fraction #
Question 2 – take A280 Screen 2.
Protein Purification Tutorial
Question 2. Which fractions contained the desired enzyme?
Fraction
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
#
A280
EnzAssay
Results
•
Enzyme activity can be
determined by performing an
enzyme assay on each fraction
that contains protein.
•
Notice the results of the enzyme
assay. The highest activity
corresponds to one of the peaks.
0 0 0 2 5 2 0 0 0 2 5 8 5 2 0 0 2 5 2 0
Need to substitute values for the colored
spots since we are switching to an absorbance
based assay.
A280
Fraction #
Now we can have them discard
tubes that don’t have enzyme
activity.
Question 2 – take A280 Screen 3.
Protein Purification Tutorial
Question 2. Which fractions contained the desired protein?
Fraction
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
#
A280
•
Enzyme activity can be
determined by performing an
enzyme assay on each fraction
that contains protein.
•
Notice the results of the enzyme
assay. Notice that the highest
activity corresponds to one of the
peaks.
0 0 0 2 5 2 0 0 0 2 5 8 5 2 0 0 2 5 2 0
EnzAssay
Results
•
A280
Fraction #
Discard the fractions that don’t
contain protein by clicking on the
tubes that don’t contain protein.
POOL fractions – screen 1
Protein Purification Tutorial
Combine (pool) the fractions with activity.
1. NEXT We want to pool the
fractions that have enzyme
activity.
Fraction
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
#
It may be useful to consider more than just the activity of a
fraction. Specific activity is a measure of the amount of
enzyme activity per amount of protein (units/mg). The
higher the specific activity, the higher the purity. When
pooling fractions, judgement is needed as to whether to
optimize yield or specific activity.
Fraction #
Pool
fractions
Protein Purification Tutorial
POOL fractions – screen 2
Combine (pool) the fractions with protein and activity.
1. The fractions are pooled together.
How do we monitor the progress of
the purification?
Fraction #
Assess purity – screen 1
Protein Purification Tutorial
1. Look at it on a gel. Even when
Question 3: Is this pooled sample pure? How do you monitor
the progress?
overloaded,
if only one band is
Standards | Crude Ext. | Pooled fractions
Purification Table
Purification Table
Procedure
Fraction vol
(ml)
Crude cellular
extract
1400
Separation
method 1
90
visible, it is likely to be pure and a
monomer, a homo-dimer, or a
homo-multimer. If not a single
band, additional bands that purify
in parallel, will remain proportional
throughout the purification. If a
band co-purifies, it is likely to be a
subunit for the enzyme.
2.
the specific activity
by
TotalCalculate
Prot
Activity
Specific activity
(mg)doing a careful
(units) quantitative
Units/mg
assay
for enzyme
activity/total10protein.
10000
100000
400
80000
200
Results:
1.
Gel shows more than one band.
Since the sample is not pure, you must pass pooled sample through another separation technique
Another separation
Assess purity – screen 2
Protein Purification Tutorial
Question 3: Is this pooled sample pure? How do you monitor the progress?
1. Look at it on a gel. A monomer
should have one band.
2. Calculate the specific activity by
doing a careful quantitative assay
for enzyme activity/total protein.
Standards | Crude Ext. | Pooled fractions
Purification Table
Purification Table
Procedure
Fraction vol
(ml)
Total Prot
(mg)
Activity
(units)
Specific activity
Units/mg
Crude cellular
extract
1400
10000
100000
10
Separation
method 1
90
400
80000
200
Separation
method 2
8
4
60000
15000
Results:
1.
Gel shows one band
2.
Specific activity is 15000. Looks good
Your protein seems pure. YOU’RE DONE!!!.
Intro – table of common techniques
Protein Purification Tutorial
Table of common methods of protein purification
Property
solubility
Methods
Precipitation with
ammonium sulfate
(salting out)*
The 3 separation techniques of chromotography
size
Size-exclusion
chromotography
Isoelectricpoint
(charge)
Ion exhange
chromatography
binding to small
molecules
Affinity
chromatography
Ammonium sulfate precipitation is cheap, easy, and accommodates large sample sizes.
It is commonly one of the first steps in a purification scheme.
Protein Purification Tutorial
Gel filtration 1 -basis
Here’s our sample mix of proteins.
Our goal is to purify protein #….
60 Kd
20 Kd
20 Kd
5 Kd
Low pI (6)
Low pI (7)
Medium pI (7)
Hi pI (8)
•
Gel filtration column
chromotography separates
proteins on the basis of size.
•
•
We will start with 4 proteins.
You will want to purify the “yellow
one”
Protein Purification Tutorial
Protein Purification Tutorial
Gel filtration 2 - close up of beads
•
The matrix of a size-exclusion
chromatography column is porous
beads.
Run column
Need two pore sizes (other size
bigger than black proteins, smaller
than existing pores.)
Protein Purification Tutorial
Gel filtration 3 - run close up of column
•
The matrix of a gel filtration
column are beads with pores.
•
The large green proteins can’t fit
in pores so flows faster.
The red/yellow medium sized
proteins get trapped in the pores.
The black small proteins stay
trapped in pores longer.
•
•
We’re wondering how this will work in
animation. The black can permeate all
pores and the space between beads, the
yellow and red can permeate the space
between beads and larger pores, the
green will be restricted to the space
between beads.
Protein Purification Tutorial
Gel filtration 4 - zoom out
Click on the peak that represents the
protein of the largest molecular
weight?
Be sure to keep in mind that the colors will either
be in the column or in the tubes, not both!
Tubes march in from left
A280
Fraction #
Protein Purification Tutorial
•
Gel Filtration 5.
•
Many columns are commercially
made. Here are some examples.
This could be moved to the earlier view of
the porous beads.
Fig 1.1. Scanning electron micrograph
of an agarose gel. Magnification x 50,000.
Ref. Anders S. Medin,PhD Thesis,
Uppsala University 1995.
Sephadex./
ION –EXCHANGE 1
Protein Purification Tutorial
60 Kd
20 Kd
20 Kd
5 Kd
Low pI (6)
Low pI (7)
Medium pI (7)
Hi pI (8)
•
Ion-exchange column
chromotography separates
proteins on the basis of charge.
•
•
•
We will start with 4 proteins.
pH 7.2
pos charged column
Need to include a slide on how to determine the charge on a protein,
given its pI and the pH.
Ion Exchange 2 – loaded proteins
Protein Purification Tutorial
•
•
pos
The matrix of an ion exchange is
positively charged.
What do you think will happen?
pos
Run column
pos
pos
pos
pos
Ion Exchange 3 –column run
Protein Purification Tutorial
•
pos
pos
pos
pos
pos
pos
•
The matrix of an ion exchange is
positively charged.
Only the pos charged proteins run
through the pos charged column.
The others “stick” to the column.
These beads are porous, too, so you can show the
proteins moving right through the beads in the
animation, ifyou like.
Protein Purification Tutorial
Ion Exchange 4- zoom out
Only the POS charged proteins run
through the column.
How can we elute the other proteins?
20
1
Tubes march in from left
A280
Fraction #
Protein Purification Tutorial
Ion Exchange 5 - zoom out
Increase the salt.
Tubes march in from left
A280
Fraction #
Add salt
Ion Exchange 6 – after salt
Protein Purification Tutorial
Increase the salt.
Add salt
What protein will come of the column
next?
-
-
---
Don’t show the charges on the color spots students should figure this out on their own!
Tubes march in from left
A280
Fraction #
Ion Exchange 7 – after salt
Protein Purification Tutorial
Increase the salt.
What protein(s) will come of the
column next? +
----Feedback statement.
Run column
Tubes march in from left
A280
Fraction #
Ion Exchange 8 – after salt
Protein Purification Tutorial
Red and yellow will have the same
neg charge and will co-elute.
1.5
Tubes march in from left
A280
Salt
concentration
0.0
Fraction #
Ion Exchange 9 – Increase salt conc. Again.
Protein Purification Tutorial
Add salt
Increase the salt concentration
1.5
Tubes march in from left
A280
Salt
concentration
0.0
Fraction #
Ion Exchange 10 – rum column prompt.
Protein Purification Tutorial
Run the column.
Run column
1.5
Tubes march in from left
A280
Salt
concentration
0.0
Fraction #
Ion Exchange 11 – results.
Protein Purification Tutorial
Run the column.
Run column
1.5
Tubes march in from left
A280
Salt
concentration
0.0
Fraction #
Ion Exchange 12 – results.
Protein Purification Tutorial
Notice that 2 of the proteins eluted at
the same time. Why?
Is our protein pure? We were
supposed to purify the red one
.
1.5
Tubes march in from left
A280
Salt
concentration
0.0
Fraction #
Protein Purification Tutorial
•
Affinity Chromotography
Affinity Chromotography.
•
See notes below
Protein Purification Tutorial
•
Monitoring progress.
Monitoring progress.
• We need some info on the SDS
page and the specific activity.
• See Jim’s hand written notes as a
guide.
Place final table here - we can include the thing plot with protein conc going
down, enzyme amount remaining constant, and specific activity on the rise.