Exp. 7 IonEx
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Biochemistry 3723
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Exp.7 Ion Exchange Chromatography
Sept. 18/23, 2002
Objectives:
1. to understand the principles behind the common types of column chromatography (gel
filtration, ion exchange, hydrophobic interaction, affinity)
2. to appreciate the power of these types of chromatography in protein purificaiton
3. to gain experience preparing, loading running and assaying columns
I. Column Chromatography-- Various types of chromatography can be used to separate proteins
in further protein purification
A. General requirements for all Columns:
1. Pouring column--almost an art form. Must be evenly poured, no air pockets, no fines
2. Size -from Pasteur pipet size to several stories tall. Temperature control
3. Loading of sample-- w/o disturbing column. Volume of sample important in some
types, not others.
4. Running column/eluting peaks--many considerations
5. Monitoring material on column.
B. Gel Exclusion--Will talk about another day
1. Separate based on SIZE of protein--See diagram below
a. large proteins cannot enter pores of beads. Elute in void volume
b. medium sized molecules enter some of pores (how much/many depends on size of
molecules) separate by size-- larger elute before smaller. Depends on size of
bead pores--different ranges. Sephadex G-5-G-200 for example.
c. smallest molecules totally included-- elute in total volume.
2. How to calculate MW-- log(MW) vs. elution volume
a. Run standards of known MW through column. Determine elution volume
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b. Prepare standard curve of log MW vs. elution volume
c. Then run sample on SAME column.
d. Properly maintained column can be used over and over again.
3. important volume limits
a. void volume--minimum volume in which material elutes from column if not
"included" in the beads at all
b. total volume--volumen in which all sample elutes from column.
c. Separation achieved only for samples eluting in volumes between these two.
C. Ion Exchange: What we are doing this week
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1. Types of ion exchange resins
a. anion exchangers--mainly based on RxN+ resins
i. strong:--R4N+--always charged--bind the tightest
ii. weak:-R3NH+-can be titrated to R3N-generally bind less tightly
b. cation exchangers--Sulfonic acid, carboxylic acid, phosphate, etc.
i. strong--Sulfonic acid--ionized at all “reasonable” pHs
ii. weak-- carboxylic acid, etc.--can be titrated so resin not charged
2. Choosing & loading conditionsa. usually want resin with charge opposite the net charge of protein of interest so that
protein will stick to column (so will some others). (Alternatively, bind only
proteins you want to get rid of).
b. Chose buffer and pH so desired protein sticks. Hopefully many other proteins
have same charge as resin and will not stick. Note: want buffer salt to be same
charge as resin, so Tris is good for anion exchangers, not for cation exchange
resins. buffer concentration not too high
3. Elution conditions
a. Change pH to change ionic form of protein OR resin (so they no longer attract)
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b. Compete with salt. (This doesn’t change charge attraction, but since is an
equilibrium process, great excess of salt will compete for sites on resin, knocking
off protein. What salt to use? Usually NaCl or KCl. But depends on interactions
with protein.
c. Gradient vs. Step elution
i. Gradient is gradual change of conditions, gives selective elution of proteins as
salt concentration or pH changes
ii. Step Elution is sudden change. Quickly knocks off whatever will come off.
Not as selective, but doesn’t dilute eluant as much.
D. Other chromatographic separations of proteins--Will Talk about another day
1. Chromatofocusing--Based on pI of protein--a type of ion exchange.
2. Affinity Chromatography--will discuss and do next week
3. Hydrophobic Interaction Chromatography
a. Uses a resin that is hydrophobic: Long carbon chain (C8-C18) or phenyl residue
attached to inert matrix. Hydrophobic patches on protein will bind to resin
b. Load protein onto resin in high salt, where they are least aqueous soluble (the
same principle as salting out of ammonium sulfate precipitation).
c. Elute by reducing salt gradient, which increases protein solubility in buffer used
as eluent, thus allowing them to detach from resin and elute.
d. This is similar to reverse phase chromatography--see below
E. HPLC--High Performance Liquid Chromatography--Talk About LATER
1. Refers to the equipment used
2. Gel Exclusion, Ion Exchange, Affinity, etc. can all be done by HPLC
3. Most often use Reverse Phase HPLC, which is similar to hydrophobic interaction
chromatography -with hydrophobic stationary phase, hydrophilic mobile phase
(acetonitrile or methanol in water commonly used).
F. Today's experiment--Use anion exchange resin (+ resin binds – proteins)
1. Resin: Bio-Rad Macro-Prep High Q. (R4N+ resin).
2. Buffer: 0.05 M Tris, pH 7.5: GOT has a pI about 5.4, so exists as an anion at pH 7.5
and will bind to quaternary ammonium resin
3. Loading sample--in 0.05 M Tris, pH 7.5.
4. Eluting. After 15 min wash at 1.0 ml/min in Tris, do linear gradient 0 to 0.4 M NaCl
(in 0.05 M Tris, pH 7.5) over 100 minutes: Cl– ions compete for binding sites on
resin, cause proteins to elute.
5. Note: you do not need to collect for the entire 105 min.
6. Assay (on Wednesday) PROTEIN containing fractions for GOT activity
III. Practical for today
A. Before you do anything else
1. Thaw GOT-1 sample (after dialysis)
2. Save 50 µl in microfuge tube, frozen for later assays. Measure volume of rest
3. Fill chromatography rack with clean test tubes
B. The equipment
1. BioRad Liquid Chromatography Econoline-Pump controllers
2. Transparency of panel
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C. The chromatography--follow instructions exactly. When in doubt, ask!
1. The instrumentation-reservoir of buffers, pump, column, UV spec, recorder and
fraction collector. Will walk through in class.
2. The program:
a. Buffer A (0.05 M Tris) for 15'
b. Linear gradient: 0-100% Buffer B (0.05 M Tris, 0.4 M NaCl) over 90' (15-105')
D. The results: Save all fractions, frozen Assay next lab.
0.4 M
Protein
A280
NaCl
0.0 M
15
minutes
105
IV. Problems
1. What kind of chromatography resin would you use to separate a mixture of amino acids
containing glutamic acid, serine, histidine, and lysine? What conditions would you use to
load the amino acids (choose conditions under which at least three of the amino acids will
stick to the resin). How can you then sequentially remove the amino acids from the
column? (And yes, I expect you to know enough about the amino acids to answer this
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question). Show an elution profile indicating the order in which the amino acids elute.
How would you follow the elution?
Solution: To do this you need to know something about the ionic state of the different
amino acids as a function of pH. Glutamic acid has 2 -COOH and one NH3+ groups. It
has a pI of about 3. Serine is aliphatic and has a pI of about 6. Histidine is a basic amino
acid and has a pI of around 7.6. Lysine is also basic and has a pI around 9.7. I would use
an anion exchange column at a pH of 8 - 9, for example MacroQ, with pH 8.3 Tricine
would work well. Under these conditions Glu, Ser, and His will all carry negative
charges and Lys will by positive. I would load the sample in pH 8.3 Tricine (50 mM) and
wash with 50 mM Tricine until the Lys came off the column. I would then use a linear
gradient of decreasing pH (made by mixing, for example, 50 mM Tricine, pH 8.3 with 50
mM Succinate, pH 5.0) to elute the other amino acids. As the pH decreases the amino
acids will elute in the order His>Ser>Glu. To follow the elution I would spot a small
amount of each fraction collected on a piece of filter paper and spray with ninhydrin
reagent, which will turn the spots blue if there is amino acid in the fraction. The elution
profile I would expect would look something like this:
Another way to separate the amino acids by ion exchange would be to bind them to a
cation exchange resin (Carboxymethyl cellulose for example) at a pH of about 5. Glu
won't bind to the resin under these conditions, but the other three will. Elute with a
gradient of increasing pH. In this case the elution order will be Glu>Ser>His>Lys.
2. What type of chromatography resin would you use to separate a mixture of the following
proteins in a bovine sample: Insulin (5.7 kDa), Myoglobin 17.0 kDa, BSA (64.5 kDa),
and Glutamate dehydrogenase (333 kDa)? How would you load the samples onto the
column, how would you then elute the different proteins, and what would you expect the
elution profile to look like? What technique would you use to “see” the proteins as they
came off the column?
Solution: This separation is best done using a gel filtration type of column. You want a
resin of a pore size that will allow these samples to separate. Looking in your book (p.
82), there are many resins you can use. Sephadex G-100 (range 4-150 kDa), for
example, will completely exclude the GDH, completely include the Insulin, and partially
include BSA and Mb. You want to load the sample in a concentrated form, with a
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volume not more than about 5% of the total volume of the column for best separation.
Any buffer in which the proteins are stable will work, for example, Tris, pH 7.8. Load
the proteins onto the column in the buffer continue to wash with the same buffer to elute.
To monitor the elution, simply measure the A280 of the eluent. The proteins will elute in
the order: GDH (in void volume)>BSA>Mb>Insulin.