Bốn kỹ thuật sắc ký (chromatographic techniques)

1.Sắc ký trao đổi ion [Ion Exchange Chromatography

(Charge specific)]

- Hydroxyapatite
-Salt tolerant ligands

2. Sắc ký lọc gel [Size Exclusion (Gel Filtration)

Chromatography]
- Size/shape dependent
3. Sắc ký ái lực [Affinity Chromatography (Biological
Interaction)]
– Protein A (Protein G and others)
– IMAC (His/surface specific)
– Specific affinity resins (lectines, dyes…)
– Immunospecific media

4. Sắc ký tương tác kỵ nước [Hydrophobic Interaction

Chromatography (Hydrophobic Patches)]
- Mixed Mode
- Hydrophobic charge induced chromatography
 What does „strategy“ mean?

Combination of different types of chromatography

• Due to the complexity of mixtures of biological materials, often several
steps are necessary to produce a homogeneous protein.
• Successive separation steps must be synchronized optimally to avoid
unnecessary re-buffering or dialysis steps.
• Otherwise the recovery of the biological activity and the mass recovery,
which both depend on the number of purification steps, will be reduced.
• For example, starting with a high salt concentration in HIC is in contrast
to ion exchange, where one has to start with very low ionic strength.
 Lưu ý trong sắc ký
• Giữ được hoạt tính sinh học:
– Không sử dụng dung môi có pH quá cao hoặc thấp,
– Nồng độ muối thích hợp,
– Thời gian tách ngắn để tránh tối thiểu quá trình phân hủy
protein
– Nhiệt độ thấp
Basic Considerations
• Cell disruption method
• Homogeneity of the source material
• Stability of the target protein
• pH
• Buffer (Stabilisators)
• Temperature (4°C)
• Proteinases (proteolysis, rapid decrease of biol. activity)
• Main contaminating compounds
• Refolding?
ProteinPurificationTutorial Intro–tableof commontechniques

B¶ngc¸cph­¬ngph¸pth­êngdïng®
ÓtinhchÕprotein
• C¸cb­íctinhchÕ cãxuthÕ b¶o® ¶mcho
Property Methods proteinë tr¹ngth¸i tùnhiÖ n. M Æcdï thÕ
m étsèproteincã thÓ t¸i tùnhiÕ n(re-
solubility Precipitationwith natured), hÇuhÕtlµkh«ngthÓ !
am m oniumsulfate
(saltingout)*
• §Ó tinhchÕproteintronghçnhîp, cãrÊ t
nhiÒuph­¬ngph¸pkh¸cnhauthÝ chhîp
Size/ shape Size-exclusion chotõnglo¹i protein. Chóngkh¸cnhauvÒ
chrom otography

– TÝ
nhbÕ
nnhiÖ
t*
Isoelectricpoint Ionexhange
(charge) chromatography

bindingtosmall Affinity
m olecules chrom atography

*KÕt tñaammoniumsulfatelµrÊ
t rÎ, dÔkiÕ
mvµphï hîpchoc¸cmÉ
ulín. Nãth­ ênglµb­ íc
®Ç
utiªntrongs¬® åtinhchÕ.
*ChohÇ uhÕtc¸cqu¸ tr×nhtinhchÕ
protein, tÊtc¶c¸cb­ ícthùchiÖnë
~5°C® Ó lµmgi¶nsùbÊ tho¹t.
Protein Purification Tutorial Intro side bar

H×nh ¶nh gel protein ví i c¸ c ®­ êng ch¹ y

chØra tr×nh tù c¸ c b­ í c tinh s¹ ch

B¶ng tinh chÕ

C¸ c b­ í c ThÓtÝch ph©n Protein tæng Ho¹ t tÝnh Ho¹ t tÝnh riª ng Purification
Yield
®o¹ n (ml) (mg) (unit) Unit/mg factor

Crude cellular 1400 10000 100000 10

1 100%
extract
Size-exclusion 90 400 80000 200 80%
20
Ion exchange 80 100 60,000 600 3 75%

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).
 Intro – flow chart. Purification is a multi step
Protein Purification Tutorial procedure

Tinh chÕlµ mét c«ng ®o¹ n cã nhiÒu b­ í c.

MÉu
Kü thuËt t¸ ch

LËp l¹ i ví i c¸ c kü
Fractionation
thuËt kh¸ c ®Õn tËn
khi tinh khiÕt

No
Assay total protein
Assay enzyme activity

No yes Combine Pure?

Set aside Is there activity? Monitor purity
Fractions

yes

Prepare for analytical technique

Protein Purification Tutorial Intro – first steps.

• First steps
– 1. Nguån. Mét nguån tèt lµ ph¶i rÎ
vµ s½n cã. NhiÒu protein cã nhiÒu
trong c¸ c m« ®Æc biÖt, vÝdô nh­
hemoglobin trong m¸ u. V×lý do
nµy mµ nhiÒu m« lµ nh÷ng nguån
tuyÖt vêi cho protein cña chóng ta.
– 2. Thö nghiÖm: hÇu hÕt c¸ c thö
nghiÖm lµ c¸ c ph¶n øng ho¸
hâcóc t¸ c nhê c¸ c ho¹ t tÝnh ®Æc
tr­ ng. C¸ c protein kh«ng cã ho¹ t
tÝnh th­ êng ®­ î c thö nghiÖm nhê
SDS polyacrylamide gel.
Protein Purification Tutorial Assay – develop an assay

• First steps – Ph¸ t triÓn thö nghiÖm

1. Mét thö nghiÖm cho mét enzyme lµ

mét ph­ ¬ng ph¸ p ®Óx¸ c ®Þnh ho¹ t
tÝnh cña nã.

Tõ thö nghiÖm nµy ®­ î c lËp l¹ i nhiÒu lÇn,

nã rÊt quan träng nh­ ng nã lµ b­ í c
®¬n gi¶n. Th«ng th­ êng ho¹ t tÝnh
enzyme ®­ î c hiÓn thÞsù thay ®æi vÒ
sù hÊp thô mµ cã thÓ®o ®­ î c nhê
spectrophotometer. VÝdô thö
ribonuclease ®o sù thay ®æi vÒsù hÊp
thô ®i cï ng ví i sù ph©n huû RNA
thµnh c¸ c ribonucleotide.
Protein Purification Tutorial Intro – Preparing the sample (Crude Extract)

First steps: ChuÈn bÞmÉu – triÕt th«.

Protein tõ c¸ c tÕbµo hoÆc m«

N­ í c næi ví i
protein hoµ tan
Ph¸ vì tÕbµo,
m« hoÆc c¬ quan

NghiÒn,
®ång ho¸ ,
Siª u ©m,
¸ p lùc,
TÕbµo vi thÈm thÊu KÕt l¾ng cã c¸ c tÕbµo, organelles,
sinh vËt mµng vµ c¸ c protein mµng
hoÆc m«
Protein Purification Tutorial Segway to Chromotography

• L­ u ý: Ví i môc ®Ých ®Ót¸ ch ®ñ l­ î ng

protein, chóng ta cÇn l­ î ng m« ban
®Çu hµng kilogram. Khèi l­ î ng nµy
tèt nhÊt cho viÖc sö dông kÕt tña
(nh­ .kÕt tña ammonium sulfate). Sau
®ã tinh chÕ, c¸ c cét lí n cã thÓsö dông
tõ gram ®Õn milligram. Khèi l­ î ng
®Óchay gel lµ microgram.
 Intro – flow chart. Purification is a multi step
Protein Purification Tutorial procedure

Tinh chÕlµ mét c«ng ®o¹ n gåm nhiÒu b­ í c.

Sample
Separation
technique

Repeat with another

Fractionation
separation
technique until pure

No
Assay total protein
Assay enzyme activity

No yes Combine Pure?

Set aside Is there activity? Monitor purity
Fractions

yes

Prepare for analytical technique

Protein Purification Tutorial Over view of the apparatus

S¾c ký cét (Column Chromotography) – tr­ êng hî p chung

– Cét thuû tinh (Glass column)
– B×nh chøa (Reservoir)
– ChÊt nhåi r¾n (Solid matrix – beads)
– Dung dÞch protein (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
• H×nh ¶nh hån hî p protein n»m
ë trª n bÒmÆt
Note - this should be shown as
a single band (possibly brown or
striped with four colors)
Load sample (4 protein mix)
DÞch chiÕt th« ®­ î c ®Ótrª n ®Ønh cña chÊt
nhåi r¾n (solid matrix). (trong tr­ êng
hî p nµy chóng ta sö dông hçn hî p 4
protein, hiÓn thÞb»ng c¸ c mµu kh¸ c
nhau.)
• (As the animation proceeds)
Protein chuyÓn ®éng ë c¸ c tèc ®é kh¸ c
nhau qua chÊt nhåi dùa trª n c¸ c tÝnh
chÊt cña protein vµ kiÓu chÊt nhåi cét.
Protein Purification Tutorial Collect fractions.

• Sù ph©n ®o¹ n • V×cét t¸ ch protein ë d¹ ng hån hî p,

“effluent” nhá xuèng c¸ c èng ph©n
®o¹ n mµ nã chuyÓn ®éng ví i mét vËn
tèc ®Æc tr­ ng. C¸ c èng nµy gäi lµ c¸ c
ph©n ®o¹ n (fraction).

ë ®©y chóng ta cã 20 èng. M¸ y thu ph©n

®o¹ n ë hÇu hÕt c¸ c phßng thÝnghiÖm
th­ êng cã kho¶ng 75-200 èng.

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—

• Trong thùc tÕ, protein kh«ng cã mµu 1. Lµm thÕnµo chóng ta biÕt ®­ î c ph©n
v×vËy chóng ta sÏ cã 3 c©u hái: ®o¹ n cã protein?

2. Ph©n ®o¹ n nµo cã protein mong

muèn?

3. Chóng ta ®¸ nh gi¸ ®é tinh khiÕt nh­

thÕnµo?

???
Protein Purification Tutorial Question 1 – take A280 Screen 1.

Question 1. Lµm thÕnµo chóng ta biÕt ®­ î c ph©n ®o¹ n cã protein?

• Tæng sè protein cã thÓdù ®o¸ n nhê
®o sù hÊp thô trª n m¸ y so mµu ë 280
nm. C¸ c amino acid th¬m hÊp thô
¸ nh s¸ ng ë b­ í c sãng lµm cho tÊt c¶
Fraction protein cã hÊp thô ë 280nm. NhiÒu
#
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
m¸ y ph©n ®o¹ n ®o A280 khi ch¹ y cét
.

Take A280
otein Purification Tutorial Question 1 – take A280 Screen 2 .

Question 1. Lµm thÕnµo chóng ta biÕt ®­ î c ph©n ®o¹ n cã protein?

• Tæng sè protein cã thÓdù ®o¸ n nhê
®o sù hÊp thô trª n m¸ y so mµu ë 280
nm. C¸ c amino acid th¬m hÊp thô
¸ nh s¸ ng ë b­ í c sãng lµm cho tÊt c¶
protein cã hÊp thô ë 280nm. NhiÒu
n m¸ y ph©n ®o¹ n ®o A280 khi ch¹ y cét
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
• C¸ c gi¸ trÞcã thÓdùng ®å thÞso ví i
0 0 0 2 5 2 0 0 0 2 5 8 5 2 0 0 2 5 2 0 sè ph©n ®o¹ n gäi lµ elution profile.

Plot values
Protein Purification Tutorial Question 1 – take A280 Screen 3 .

Question 1. Lµm thÕnµo chóng ta biÕt ®­ î c ph©n ®o¹ n cã protein?

• Protein tæng cã thÓdù ®o¸ n nhê ®o
hÊp thô ë 280 nm trong m¸ y so mµu.
• C¸ c gi¸ trÞcã thÓdùng ®å thÞso ví i
sè ph©n ®o¹ n gäi lµ elution profile.

Fraction
#
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 • L­ u ý c¸ c peak trª n ®å thÞ. Nã hiÓn
0 0 0 2 5 2 0 0 0 2 5 8 5 2 0 0 2 5 2 0
thÞë ph©n ®o¹ n nµo cã protein.
A280

Peaks

A280

Fraction #
Protein Purification Tutorial Question 1 – take A280 Screen 4 .

Question 1. How do we know which fractions contain protein?

• 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
Fraction called an elution profile.
#
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
0 0 0 2 5 2 0 0 0 2 5 8 5 2 0 0 2 5 2 0
A280 • Notice the peaks on the graph.
These indicate where the fractions
Peaks are that contain protein.

A280

Fraction #
Protein Purification Tutorial Question 2 – take A280 Screen 1 .

Question 2. Ph©n ®o¹ n nµo cã protein mong muèn?

• Ho¹ t tÝnh enzyme cã thÓthÓx¸ c ®Þnh
nhê thùc hiÖn thö enzyme trª n mçi
ph©n ®o¹ n mµ cã 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
 Protein Purification Tutorial Question 2 – take A280 Screen 2.

Question 2. Ph©n ®o¹ n nµo cã protein mong muèn?

• Ho¹ t tÝnh enzyme cã thÓx¸ c ®Þnh nhê
thùc hiÖn thö nghiÖm enzyme trª n
mçi ph©n ®¹ on cã protein.

• L­ u ý c¸ c kÕt qu¶ thö nghiÖm

Fraction
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 enzyme. Ho¹ t tÝnh cao nhÊt t­ ¬ng øng
#
ví i 1 trong c¸ c peak.
0 0 0 2 5 2 0 0 0 2 5 8 5 2 0 0 2 5 2 0
A280
EnzAssay
Results
Need to substitute values for the colored
spots since we are switching to an absorbance B©y giê chóng ta cã thÓ
based assay.
bá c¸ c èng mµ kh«ng cã
enzyme.
A280

Fraction #
 Protein Purification Tutorial Question 2 – take A280 Screen 3.

Question 2. Ph©n ®o¹ n nµo cã protein mong muèn?

• 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 • L­ u ý c¸ c kÕt qu¶ cña thö enzyme.
#
Ho¹ t tÝnh cao nhÊt t­ ¬ng ®­ ¬ng ví i
0 0 0 2 5 2 0 0 0 2 5 8 5 2 0 0 2 5 2 0
A280 mét trong c¸ c peak.
EnzAssay
Results

• Lo¹ i bá c¸ c ph©n ®o¹ n kh«ng cã

A280 protein.

Fraction #
Protein Purification Tutorial POOL fractions – screen 1

KÕt hî p c¸ c ph©n ®o¹ n cã ho¹ t tÝnh.

1. TiÕp theo chóng ta gom c¸ c ph©n

®o¹ n cã ho¹ t tÝnh enzyme.

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. Pool
fractions

Fraction #
Protein Purification Tutorial POOL fractions – screen 2

Gom c¸ c ph©n ®o¹ n cã protein vµ ho¹ t tÝnh.

1. C¸ c ph©n ®o¹ n ®­ î c gom l¹ i ví i nhau.

How do we monitor the progress of the

purification?

Fraction #
Protein Purification Tutorial Assess purity – screen 1

1. Xem trª n gel. NÕu chØcã mét

Question 3: MÉu gom ®ã cã tinh khiÕt
kh«ng?
band, nã cã thÓtinh khiÕt vµ lµ
Standards | Crude Ext. | Pooled fractions mét monomer, homo-dimer, hoÆc
homo-multimer. NÕu cã nhiÒu
band th×sù tinh chÕcã thÓch­ a
s¹ ch hoÆc c¸ c subunit cña
enzyme.
Purification Table 2. TÝnh ho¹ t ®é riª ng th«ng qua x¸ c
Purification Table ®Þnh ho¹ t tÝnh enzyme/protein
Procedure Fraction vol Totaltæng.
Prot Activity Specific activity
(ml) (mg) (units) Units/mg

Crude cellular 1400 10000 100000 10

extract
Separation 90 400 80000 200
method 1

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
Protein Purification Tutorial Assess purity – screen 2

Question 3: mÉu enzyme tinh chÕcã tinh khiÕt hay kh«ng?

Standards | Crude Ext. | Pooled fractions 1. Quan s¸ t nã trª n gel. Monomer sÏ cã

one band.
2. TÝnh ho¹ t tÝnh riª ng nhê tû sè ho¹ t
tÝnh enzyme tæng/ tæng sè protein.

Purification Table
B­ í c thùc ThÓtÝch ph©n Protein tæng H¹ t tÝnh tæng Ho¹ t tÝnh riª ng
hiÖn ®o¹ n (ml) (mg) (units) Units/mg
Crude cellular 1400 10000 100000 10
extract
Separation 90 400 80000 200
method 1
Separation 8 4 60000 15000
method 2

Results:
1. Gel shows one band
2. Specific activity is 15000. Looks good

Your protein seems pure. YOU’RE DONE!!!.

B¶ng c¸ c ph­ ¬ng ph¸ p th«ng dông tinh chÕprotein

TÝnh chÊt Ph­ ¬ng ph¸ p

KÕt tña b»ng
TÝnh tan
ammonium sulfate
(salting out)*

KÝch th­ í c S¾c ký läc gel

§ iÓm ®ång ®¼ng S¾c ký trao ®æi ion

®iÖn (®iÖn tÝch)

KÕt hî p ví i c¸ c S¾c ký ¸ i lùc

ph©n tö nhá

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 #….
• S¾c ký cét läc gel t¸ ch protein dùa
trª n kÝch th­ í c.

• Chóng ta sÏ b¾t ®Çu ví i 4 protein.

• Chóng ta muèn tinh chÕ“yellow one”

60 Kd 20 Kd 20 Kd 5 Kd

Low pI (6) Low pI (7) Medium pI (7) Hi pI (8)

Protein Purification Tutorial Gel filtration 2 - close up of beads

• C¬ së chÝnh cña kü thuËt s¾c ký cét

läc gel lµ lç rçng cña chÊt nhåi cét.

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

• Gi¸ thÓcña cét läc gel lµ c¸ c h¹ t cã

lç.

• C¸ c protein lí n xanh kh«ng võa ví i

c¸ c lç v×vËy nã chÈy nhanh h¬n.
• C¸ c protein trung b×nh red/yellow bÞ
gi÷ l¹ i trong c¸ c lç.
• C¸ c protein nhá black bÞgi÷ l¹ i trong
c¸ c lç l©u h¬n.

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./
Protein Purification Tutorial ION –EXCHANGE 1

• S¾c ký cét trao ®æi ion t¸ ch protein

dùa trª n ®iÖn tÝch.

• Chóng ta b¾t ®Çu ví i 4 protein.

• pH 7.2
• Cét tÝch ®iÖn d­ ¬ng
60 Kd 20 Kd 20 Kd 5 Kd

Low pI (6) Low pI (7) Medium pI (7) 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

• Gi¸ thÓcña trao ®æi ion cã tÝnh

®iÖn d­ ¬ng.
pos • Chóng ta nghÜc¸ i g×sÏ xÈy ra?
pos

Run column
pos

pos

pos

pos
Protein Purification Tutorial Ion Exchange 3 –column run

• Gi¸ thÓcña trao ®æi ion cã tÝnh

®iÖn d­ ¬ng.
pos • ChØc¸ c protein tÝch ®iÖn d­ ¬ng ch¹ y
qua cét tÝch ®iÖn d­ ¬ng. C¸ c protein
pos
kh¸ c bÞgi÷ l¹ i trong cét.

pos
These beads are porous, too, so you can show the
proteins moving right through the beads in the
animation, ifyou like.
pos

pos

pos
Protein Purification Tutorial Ion Exchange 4- zoom out

ChØcã c¸ c protein tÝch ®iÖn d­ ¬ng ch¹ y

qua cét.

Lµm thÕnµo ®Óchóng ta lÊy ®­ î c c¸ c

protein kh¸ c?

20 1

Tubes march in from left

A280

Fraction #
Protein Purification Tutorial Ion Exchange 5 - zoom out

T¨ ng nång ®é muèi. Add salt

Tubes march in from left

A280

Fraction #
Protein Purification Tutorial Ion Exchange 6 – after salt

T¨ ng néng ®é muèi. Add salt

Protein nµo sÏ ra khái cét sau ®ã?
- - ---

Don’t show the charges on the color spots -

students should figure this out on their own!

Tubes march in from left

A280

Fraction #
Protein Purification Tutorial Ion Exchange 7 – after salt

+ T¨ ng nång ®é muèi.
--- Protein nµo sÏ ®i ra tr­ í c?

---

Run column

Tubes march in from left

A280

Fraction #
Protein Purification Tutorial Ion Exchange 8 – after salt

§ á vµ vµng cã cï ng ®iÖn tÝch (net charge)

vµ sÏ ®­ î c röa cï ng nhau (co-elute).

1.5

Tubes march in from left

A280 Salt
concentration

0.0

Fraction #
Protein Purification Tutorial Ion Exchange 9 – Increase salt conc. Again.

Add salt
T¨ ng nång ®é muèi

1.5

Tubes march in from left

A280 Salt
concentration

0.0

Fraction #
Protein Purification Tutorial Ion Exchange 10 – rum column prompt.

Run the column.

Run column

1.5

Tubes march in from left

A280 Salt
concentration

0.0

Fraction #
Protein Purification Tutorial Ion Exchange 11 – results.

Run the column.

Run column

1.5

Tubes march in from left

A280 Salt
concentration

0.0

Fraction #
Protein Purification Tutorial Ion Exchange 12 – results.

L­ u ý r»ng 2 protein ®i ra cï ng mét thêi

gian. T¹ i sao?
Protein cña chóng ta cã tinh khiÕt? Chóng
ta muèn tinh chÕred 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. • Chóng ta cÇn mét sè th«ng tin trª n SDS –

PAGE vµ ho¹ t tÝnh riª ng.

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.