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Protein Purification Tutorial
Protein purification tutorialTITLE 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.
Protein Purification Tutorial
Intro –Why purify proteins?
• During the last decade, the genomic sequence of many organisms has been determined. The wealth of information in these genomes has dramatically changed research. If you can identify the gene within the 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
Objectives
• In this tutorial you will:– 1
– 2
– 3
– 4
– 5
Protein Purification Tutorial
Intro – table of common techniques
Property Methods
Size / shape Size-exclusion chromotography
Isoelectricpoint (charge)
Ion exhange chromatography
binding to small molecules
Affinity chromatography
Table of common methods of protein purification
solubility Precipitation with ammonium sulfate (salting out)*
*Ammonium sulfate precipitation is cheap, easy, and accommodates large sample sizes. It is commonly one of the first steps in a purification scheme.
• Purification procedures attempt to maintain the protein in native form. Although some proteins can be re-natured, most cannot!
• To purify a protein from a mixture, biochemists exploit the ways that individual proteins differ from one another. They differ in:
– Thermal stability*
*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
Procedure Fraction vol
(ml)
Total Prot
(mg)
Activity
(units)
Specific activity
Units/mg
Crude cellular extract
1400 10000 100000 10
Size-exclusion 90 400 80000 200
Ion exchange 80 100 60,000 600
Purification Table
Add row with ammonium sulfate data. Include two colums at end called Purification factor and Yield.
Purification factor
Yield
100%
20 80%
3 75%
1
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).
Protein Purification Tutorial
Intro – flow chart. Purification is a multi step procedure
Sample Separationtechnique
Fractionation
Purification is a multi-step procedure.
Is there activity?Set aside No CombineFractions
yesMonitor purity
Assay total protein
Assay enzyme activity
Pure?
Prepare for analytical technique
yes
No
Repeat with another separation
technique until pure
Protein Purification Tutorial
Intro – first steps.
• 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.
Protein Purification Tutorial
Assay – develop an assay
1. First steps – Develop an Assay
2. 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.
Protein Purification Tutorial
Intro – Preparing the sample (Crude Extract)
Protein from cells or tissue
First steps: Preparing the sample – Crude extract.
Microbial cells or tissue
Break cells, tissue, or organ
Blender, homogenizer, sonication,pressure,psmotic Pellet with intact cells, organelles, membranes and membrane proteins
Supernatant withSoluble protein
Protein Purification Tutorial
Segway to Chromotography
• Some kind of graphic. • 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.
Protein Purification Tutorial
Intro – flow chart. Purification is a multi step procedure
Sample Separationtechnique
Fractionation
Purification is a multi-step procedure.
Is there activity?Set aside No CombineFractions
yesMonitor purity
Assay total protein
Assay enzyme activity
Pure?
Prepare for analytical technique
yes
No
Repeat with another separation
technique until pure
Protein Purification Tutorial
Over view of the apparatus
– Glass column– Reservoir– Solid matrix – beads– Solution of protein.– Effluent.– Other terms?
Column Chromotography –general case
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
Load sample (4 protein mix)
• Image of apparatus with protein mixture layered on top
• 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
Collect fractions.
• Fractionation • 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
--3 questions—
• In reality, proteins aren’t color-coded so we must ask ourselves these 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?
???
Protein Purification Tutorial
Question 1 – take A280 Screen 1.
• 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.
Question 1. How do we know which fractions contain protein?
Take A280Take A280
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Fraction#
Protein Purification Tutorial
Question 1 – take A280 Screen 2 .
• 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 can be plotted against the fraction number in is what is called an elution profile.
Question 1. How do we know which fractions contain protein?
A280
Plot valuesPlot values
0 0 0 2 5 2 0 0 0 2 5 8 5 2 0 0 2 5 2 0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Fraction#
Protein Purification Tutorial
Question 1 – take A280 Screen 3 .
• 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. How do we know which fractions contain protein?
A2800 0 0 2 5 2 0 0 0 2 5 8 5 2 0 0 2 5 2 0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Fraction#
A280
Fraction #
Peaks
Protein Purification Tutorial
Question 1 – take A280 Screen 4 .
• 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. How do we know which fractions contain protein?
A2800 0 0 2 5 2 0 0 0 2 5 8 5 2 0 0 2 5 2 0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Fraction#
A280
Fraction #
Peaks
Protein Purification Tutorial
Question 2 – take A280 Screen 1 .
• Enzyme activity can be determined by performing an enzyme assay on each fraction that contains protein.
Question 2. Which fractions contained the desired protein?
A2800 0 0 2 5 2 0 0 0 2 5 8 5 2 0 0 2 5 2 0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Fraction#
A280
Fraction #
Enz. Assay.Enz. Assay.
Fraction#
Protein Purification Tutorial
Question 2 – take A280 Screen 2.
• 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.
Question 2. Which fractions contained the desired enzyme?
A2800 0 0 2 5 2 0 0 0 2 5 8 5 2 0 0 2 5 2 0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Fraction#
A280
Fraction #
EnzAssayResults
Need to substitute values for the colored spots since we are switching to an absorbance based assay.
Now we can have them discard tubes that don’t have enzyme activity.
Protein Purification Tutorial
Question 2 – take A280 Screen 3.
• 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.
• Discard the fractions that don’t contain protein by clicking on the tubes that don’t contain protein.
Question 2. Which fractions contained the desired protein?
A2800 0 0 2 5 2 0 0 0 2 5 8 5 2 0 0 2 5 2 0
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Fraction#
A280
Fraction #
EnzAssayResults
Protein Purification Tutorial
POOL fractions – screen 1
Combine (pool) the fractions with activity.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20Fraction#
Fraction #
Pool fractions
Pool fractions
• NEXT We want to pool the fractions that have enzyme activity.
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.
Protein Purification Tutorial
POOL fractions – screen 2
Combine (pool) the fractions with protein and activity.
Fraction #
1. The fractions are pooled together.
How do we monitor the progress of the purification?
Protein Purification Tutorial
Assess purity – screen 1
1. Look at it on a gel. Even when overloaded, if only one band is 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. Calculate the specific activity by doing a careful quantitative assay for enzyme activity/total protein.
Question 3: Is this pooled sample pure? How do you monitor the progress?
Standards | Crude Ext. | Pooled fractions
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
Purification Table
Purification Table
Results: 1. Gel shows more than one band.
Since the sample is not pure, you must pass pooled sample through another separation technique
Another separationAnother separation
Protein Purification Tutorial
Assess purity – screen 2
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.
Question 3: Is this pooled sample pure? How do you monitor the progress?
Standards | Crude Ext. | Pooled fractions
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
Purification TablePurification Table
Results: 1. Gel shows one band2. Specific activity is 15000. Looks good
Your protein seems pure. YOU’RE DONE!!!.
Protein Purification Tutorial
The 3 separation techniques of chromotography
Intro – table of common techniques
Affinity chromatography
binding to small molecules
Ion exhange chromatography
Isoelectricpoint (charge)
Size-exclusion chromotography
size
MethodsProperty
Table of common methods of protein purification
solubility Precipitation with ammonium sulfate (salting out)*
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
• 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”60 Kd
Low pI (6)
20 Kd
Low pI (7)
20 Kd
Medium pI (7)
5 Kd
Hi pI (8)
Here’s our sample mix of proteins.Our goal is to purify protein #….
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 columnRun 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?
Tubes march in from left A280
Fraction #
Be sure to keep in mind that the colors will either be in the column or in the tubes, not both!
Protein Purification Tutorial
Gel Filtration 5.
• • Many columns are commercially made. Here are some examples.
Fig 1.1. Scanning electron micrographof an agarose gel. Magnification x 50,000.Ref. Anders S. Medin,PhD Thesis, Uppsala University 1995.
Sephadex./
This could be moved to the earlier view of the porous beads.
Protein Purification Tutorial
ION –EXCHANGE 1
• Ion-exchange column chromotography separates proteins on the basis of charge.
• We will start with 4 proteins.
• pH 7.2
• pos charged column60 Kd
Low pI (6)
20 Kd
Low pI (7)
20 Kd
Medium pI (7)
5 Kd
Hi pI (8)
Need to include a slide on how to determine the charge on a protein, given its pI and the pH.
Protein Purification Tutorial
Ion Exchange 2 – loaded proteins
• The matrix of an ion exchange is positively charged.
• What do you think will happen?
pos
pos
pos
pos
pos
pos
Run columnRun column
Protein Purification Tutorial
Ion Exchange 3 –column run
• 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.pos
pos
pos
pos
pos
pos
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?
Tubes march in from left
A280
Fraction #
120
Protein Purification Tutorial
Ion Exchange 5 - zoom out
Increase the salt.
Tubes march in from left
Add saltAdd salt
A280
Fraction #
Protein Purification Tutorial
Ion Exchange 6 – after salt
Increase the salt.
What protein will come of the column next?
Tubes march in from left
-
A280
Fraction #
- ---
Add saltAdd salt
Don’t show the charges on the color spots - students should figure this out on their own!
Protein Purification Tutorial
Ion Exchange 7 – after salt
Increase the salt.
What protein(s) will come of the column next?
Feedback statement.
Tubes march in from left
---
A280
Fraction #
+ ---
Run columnRun column
Protein Purification Tutorial
Ion Exchange 8 – after salt
Red and yellow will have the same neg charge and will co-elute.
Tubes march in from left
A280
Fraction #
Salt concentration
1.5
0.0
Protein Purification Tutorial
Ion Exchange 9 – Increase salt conc. Again.
Increase the salt concentration
Tubes march in from left
A280
Fraction #
Salt concentration
1.5
0.0
Add saltAdd salt
Protein Purification Tutorial
Ion Exchange 10 – rum column prompt.
Run the column.
Tubes march in from left
A280
Fraction #
Salt concentration
1.5
0.0
Run columnRun column
Protein Purification Tutorial
Ion Exchange 11 – results.
Run the column.
Tubes march in from left
A280
Fraction #
Salt concentration
1.5
0.0
Run columnRun column
Protein Purification Tutorial
Ion Exchange 12 – results.
Notice that 2 of the proteins eluted at the same time. Why?
Is our protein pure? We were supposed to purify the red one
.
Tubes march in from left
A280
Fraction #
Salt concentration
1.5
0.0
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.
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