eKsTraKsI dan
PuriFikaSi
A. Pendahuluan
• Beberapa teknik isolasi enzim secara umum
dikelompokan menjadi:
– Metode klasik berupa distilasi dan ekstraksi dengan
pelarut organik (berdasarkan sifat umum makromolekul:
pH, kekuatan ion dan kelarutan) Kurang digunakan lagi
karena kaitannya dengan kestabilan enzim
– Perbedaan sifat protein globular: Mr, muatan protein
– Interaksi spesifik dan reversibel antara enzim dengan
substrat, koenzim, ligan
Penemuan separasi enzim
Penemuan
Tahun
Pengendapan dengan alkohol
1833 (Payen & Persoz)
Adsorpsi spesifik amylase dengan
substrat insolubel
1910 (Starkenstein)
Penggunaan adsorbant untuk
pemurnian enzim
1922 (Willstarter)
Penggunaan ultrasentrifugasi
pertama kali
1923 (Svedberg & Nichols)
Kristalisasi Urease
1926 (Summer)
80 enzim terisolasi
1930
Pengenalan resin penukar ion
1935 (Adams & Holmes)
Kromatografi adsorpsi
1938-1941 (Zechmeister &
Brockman)
Metode Pengendapan terfraksi
dengan pelarut organik
1946 (Cohn)
Kromatografi dengan hidroksi
apatit
1951-1956 (Tiselius & Swingle)
Pengenalan penukar ion selulosa
1956 (Sober & Peterson)
Pengenalan sepadex dan gel
filtrasi
1959 (Porath & Flodin)
Elektroforesis Protein
1966 (Vesterberg)
Introduksi aktivasi dengan CNBr
1967 (Axen & Porath)
SDS Page elektroforesis
1967 (Shapiro)
Induksi konsep kromatografi
afinitas
1968 (Cuatrecasas)
Kromatografi hidrofob
1971 (Yon, Er El & Shaltiel)
Lebih dari 2000 enzim terisolasi
1980
PEMISAHAN MATERIAL
• Pengambilan bahan tidak larut (Removal of Insolubles). Sedikit
mengkonsentrasikan produk atau perbaikan produk. Filtrasi dan
sentrifugasi.
• Isolasi Produk. Tidak spesifik, pengambilan bahan yang mempunyai
sifat yang tersebar dibandingkan dengan produk yang diinginkan.
Konsentrasi dan kwalitas produk mulai terjadi. Adsorpsi dan
ekstraksi solven.
• Purifikasi. Teknik proses yang sangat selektif untuk menghasilkan
produk dan mengambil bahan yang tidak diinginkan serupa dengan
fungsi kimia dan sifat fisika. Khromatografi, elektrophoresis, dan
presipitasi.
• Produk akhir. Kristalisasi
B. Tahapan isolasi
• Lokasi enzim:
– Ekstraseluler (ekoenzim)
– Endoseluler (terikat pada partikel
subseluler atau membran sel)
• Hal yang perlu diperhatikan
– Sifat khas materi utama
•Bentuk (cair, padat)
•Tipe umum (animal, vegetal, mikroba)
•Struktur biologi (seluler, tisuler)
– Lokasi produk yang dicari: mitokondria,
sitoplasma, membran, dll.
• Sifat struktural dan fisiko-kimia:
–
–
–
–
Mr, struktur molekul, stabilitas
pH optimum, pI
Aktivator, inhibitor
Konstanta kinetika
• Pelepasan protein dalam bentuk cair tanpa
menghilangkan aktivitas
• Tanpa merusak struktur
– Temperatur rendah (4ºc)
– Penggunaan buffer
– Penggunaan reagen pelindung (EDTA, 2-mercaptoethanol,
substrat, dll
• Perlakuan secara cepat dan hati-hati
B.1. Tahapan Isolasi
• Materi utama sangat heterogen, maka untuk
mendapatkan enzim murni perlu tahapan isolasi
– Ekstraksi: peelepasan enzim dari sel atau bagian sel dan
didapatkan ekstrak dalam bentuk cair yang mempunyai
sifat fisiko kimia sama
– Fraksionasi: memisahkan ekstraks berdasarkan
kelarutannya guna mendapatkan kelompok molekul yang
sama (fraksi)
– Purifikasi: pemisahan fraksi lebih lanjut dengan metode
fisiko-kimia atau biospesifik untuk mendapatkan molekul
enzim lebih murni
Skema umum isolasi dan
purifikasi enzim
Materi Primer
Animal
Vegetal
Mikroba
Tahap Ekstraksi
Ekstrak total
Tahap Fraksionasi
Ekstrak kasar
Tahap Purifikasi
Enzim murni
Microbial source
• Often more stable than analogous enzyme obtained from
plant or animal tissue
• Generally Recognized As Safe (GRAS) certified microbes are
non pathogenic, nontoxic, and generally they do not produce
antibiotics
Bacteria
• Bacillus subtilis
• B. Amyloliquifaciens
• L. spesies
Fungi
•
•
•
•
•
Aspergillus sp.
Penicillium sp.
Mucor
Rhizobium
S. cerevesiae
Plant source
• Represent a traditional source of a wide range of enzymes
• Plant tissues are chosen as a source for subsequent
purification of various enzymes
Enzyme
Plant source
Ascorbate oxidase
Curcubita species
Urease
Jack bean
Bromealin
Pineapple stem/fruit
Amylase
Barley
Pectine esterase
Citrus fruits
Phytase
Wheat,rye, triticale
Animal source
• Animal tissues are a source of several enzymes of industrial
use and therapeutic use
• The other organs like stomach, placenta, heart, kidney or cells
like erythrocytes can be sources for specific enzymes
Enzyme
Animal source
Acetyl choline esterase
Bovine erythrocytes
Arginase
Beef liver
Creatine kinase
Rabbit muscle, beef heart
Aldose reductase
Beef eyes
Uricase
Porcine liver
Trypsin
Mammalian pancreas
STEP IN ENZYME PURIFICATION
Step
Enzyme
extract
Crude
Enzyme
Dilute
Enzyme
Conc.
Enzyme
Target
Treatment
I
Choosing enzyme source and
recovery
• Filtration
• Centrifugation
• Cell disruption
II
Removal of whole cells and cell
debris from enzyme extract
• Centrifugation
• Filtration
III
Removal of nucleic acids and
lipids
• Precipitation
• Nucleases
• Glass wool
IV
Concentration and primary
purification
•
•
•
•
Ultrafiltration
Precipitation
Chromatography
Dehydration
V
Final purification and quality
check
•
•
•
•
•
•
Gel filtration
Ion exchange
Affinity
Hydrophobic interaction
Chromatofocussing
HPLC
Profil Proses
Tingkatan
Produk
Proses
Kons. (g/l)
Kwalitas (%)
Pemanenan
Fermentasi
0.1 – 5
0.1 – 1.0
Pengambilan bahan
tidak terlarut
Filtrasi
1.0 – 5
0.2 – 2.0
Isolasi
Ekstraksi
5 – 50
1 – 10
Purifikasi
Kromatografi
50 – 200
50 – 80
Produk akhir
Kristalisasi
50 – 200
90 – 100
Teknik separasi dan purifikasi berdasarkan sifatnya
Ultrasentrifugasi
Dialisa
Gel Filtrasi
Ultrasentrifugasi
Sentrifugasi zonal
Kromatografi
penukar ion
Mobilitas elektroforesis
Gel Elektroforesis
Mr
Densitas
Sifat permukaan
Kromatografi
Adsorpsi
Elektroforesis
Kromatografi:
• Afinitas
•Hidrofobik
•Kovalen
Muatan
Enzim
Stabilitas
Perlakuan :
• asam-basa
• suhu
Titik isoelektrik
Elektro
dekantasi
Kelarutan
Pengendapan
Isoelektrik
Partisi
Cair-cair
Pengendapan terfraksi
dengan garam atau
pelarut organik
B.2. Kontrol Kualitas
• Enzim industrial perlu adanya kontrol kualitas yang
meliputi;
– Kemurnian enzim dalam periode waktu tertentu (aktivitas
spesifik)
– Kontrol kemurnian dengan metode fisiko-kimia;
homogenitas dan sifat karakteristiknya (Mr,
polimorfisme...)
– Uji stabilitas: resiko denaturasi, semakin murni enzim
semakin mudah terdenaturasi. Perlu dilakukan:
• Eliminasi kontaminan
• Penyimpanan pada temperatur renadah
• pH netral
Prosedur umum kontrol kwalitas enzim murni
Pengukuran :
• Aktivitas katalitik
•Protein
•Aktivitas spesifik
•Kontaminan (enzim &
lainnya
Aktivitas biologi
& Spesifik
• Elektroforesis
•Ultrasentrifugasi
•Gel Filtrasi
Pengukuran:
• Tekanan osmose
• Pengendapan dengan UF
•Difusi dan koefisien difusi
•Gel eksklusi kromatografi
Homogenitas
SDS PAGE
Mr
Enzim murni
Metode Sanger
Polimorfisme, Mr
Komposisi & Sequence
Kemasan
Stabilitas
Label Penggunaan
C. Ekstraksi
• Ekstraksi; pelepasan enzim dari sel atau
bagian sel menggunakan proses mekanik,
dan non mekanik (kimia, enzimatis, dll)
• Jaringan vegetal dan animal: penghalusan
dan homogenisasi, secara mekanik
• Sel mikroorganisme secara umum adalah
pemecahan dinding sel secara mekanik dan
non mekanik
– Kimia: alkali/asam, deterjen, osmose, EDTA
memecah bakteri gram negatif
– Enzimatis: lisosim (memutus 1-4 glukosida
peptidoglican)
Metode ekstraksi
Metode
Perlakuan
Pemecahan jaringan dan Mekanik : potong, pecah dan homogenasi
sel
Osilasi frekwensi tinggi: ultrasonikasi, turmix
Grinding
Pemecahan dan homogenisasi dengan tekanan
tinggi
Ekstraksi dengan pelarut
air
Temperatur : shock dingin
pH : shock alkali atau asam
Konsentrasi garam : shock osmosis
Efek spesifik substrat
Metode ekstraksi spesial
Pelarut organik: butanol, aseton dan pelarut lipid
Pembekuan dan pencairan
Penggunaan deterjen
Na-deoksi kolat, Tween 20, Teepol XL, Triton X100
Ekstraksi dengan enzim
Otolisis: proteolisa dan lipolisa
Penggunaan enzim pemurni (tripsin, lipase)
Pemecahan dinding mikroorganisme
• Proses mekanik:
–
–
–
–
Ultrasonikasi: sel dipecah
Pembekuan-pencairan
Penggerusan/agitasi dengan partikel gelas
Desintegrasi pada P>
• Proses non-mekanik:
– Desikasi dengan spray drying
– Liase secara kimia dan fisika
• Perlakuan alkali
• Deterjen: Na-lauril sulfat, trixtron X-100
• Shock dingin : jumlah kecil
• Shock osmotic: perubahan konsentrasi garam
– Liase Enzimatik
• Lisozim : hidrolisis beta 1,4 glukosida
• Autolisis: dengan proteolise, lipolise
D. FRAKSIONASI
D.1. FRAKSIONASI PENGENDAPAN
D.1.1. PENGENDAPAN DENGAN GARAM
• Garam yang paling banyak digunakan Amm.
Sulfat
• Prinsip:
– Protein larut dalam larutan garam pada pH
sekitar pI
– Kelarutan lebih kuat dibanding dengan kekuatan
ion dalam larutan (salting in)
– Batas kekuatan ion tertentu, kelarutan
berkurang (salting out) berkaitan dengan
terhidrasinya protein
• Daerah pengendapan bergantung pada:
– Garam yang digunakan
– Jenis proteinnya
• Kelebihan (NH4)2SO4:
–
–
–
–
Harganya murah
Kemampuan pengendapan tinggi
Kelarutannya besar, endotermik
Efek denaturasi terhadap protein rendah
• Kristalisasi garam tertentu yang
terendapkan oleh konsentrasi garam
tertentu dapat dilarutkan kembali
dengan melarutkannya pada pelarut
dengan kadar lebih rendah
D.1.2. PENGENDAPAN PADA ISOELEKTRIK
pI
Kelarutan minimum, mengendap
Pengaturan pH larutan
D.1.3. EFEK TEMPERATUR
T>Kelarutan>, s/d 40-50C
Protein Globular
Hemoglobin
Pengaturan T
Seleksi
Protein
Protein
terdenaturasi
Tidak dapat digunakan
secara industri
D.1.2. PENGENDAPAN DENGAN PELARUT
ORGANIK
Protein
Saling bergabung
Enzim
Protein
mengendap
<: konstanta dielektrikum
& kestabilan
Tambah pelarut organik
Penambahan pelarut pada
T< shg tidak mendenaturasi
• Alkohol
– Isopropanol (paling banyak digunakan) untuk enzim
ekstraseluler; amiloglukosidase
– Methanol
• Aseton dan etil eter: protein sedikit larut maka perlu
jumlah yang banyak
Variable
Extraction
Adsorption
Capacity
High
Low
Selectivity
Moderate
High
Nature of equilibrium
Often linier;
dilute solutes
independent
Usually non linier; dilute
solutes interact
Nature of operation
Steady state
Unsteady; periodic
Problems
Emulsification;
denaturation
Solids handling;
compressible packing
Electrophoresis
Electrophoresis
• Principle is to separate proteins (in
tact) on the basis of their charge and
their ability to migrate within a gel
(jello-like) matrix
• A strong electric field is applied to the
protein mixture for an extended
period of time (hours) until the
proteins move apart or migrate
Isoelectric Focusing (IEF)
Isoelectric Point (pI)
• The pH at which a protein has a net charge=0
•
Q = S Ni/(1 + 10pH-pKi)
Transcendental
equation
pKa Values for Ionizable Amno Acids
Residue
C
D
E
pKa
10.28
3.65
4.25
Residue
H
K
R
pKa
6
10.53
12.43
IEF Principles
Increasing pH
A
N
O
D
E
_
_
_
_
_
_
_
_
_
+
+
+
+
+
+
+
+
+
pI = 5.1
pI = 6.4
pI = 8.6
C
A
T
H
O
D
E
Isoelectric Focusing
•
•
•
•
•
Separation of basis of pI, not Mw
Requires very high voltages (5000V)
Requires a long period of time (10h)
Presence of a pH gradient is critical
Degree of resolution determined by slope of pH
gradient and electric field strength
• Keeps protein structure intact
• Can be scaled up to isolate mg to gms of protein
in a single “tube” gel run
Column Chromatography
Column Chromatography
• Most common (and best) approach to
purifying larger amounts of proteins
• Able to achieve the highest level of purity
and largest amount of protein with least
amount of effort and the lowest likelihood of
damage to the protein product
• Standard method for pharma industry
Column Chromatography
• Can be done either at atmospheric
pressure (gravity feed) or at high
pressure (HPLC, 500-2000 psi)
• Four types of chromatography:
– Affinity chromatography
– Gel filtration (size exclusion)
chromatography
– Ion exchange chromatography
– Hydrophobic (reverse phase)
chromatography
Affinity Chromatography (AC)
• Adsorptive separation in which the
molecule to be purified specifically and
reversibly binds (adsorbs) to a
complementary binding substand (a
ligand) immobilized on an insoluble
support (a matrix or resin)
• Purification is 1000X or better from a
single step (highest of all methods)
• Preferred method if possible
AC
Step 1: Attach ligand
to column matrix
Step 2: Load protein
mixture onto column
AC
Step 3: Proteins bind
to ligand
Step 4: Wash column to remove unwanted
material, elute later
Affinity Chromatography
• Used in many applications
• Purification of substances from
complex biological mixtures
• Separation of native from denatured
forms of proteins
• Removal of small amounts of
biomaterial from large amounts of
contaminants
Affinity Chromatography
• The ligand must be readily (and cheaply)
available
• Ligand must be attachable (covalently) to
the matrix (typically sepharose) such that it
still retains affinity for protein
• Binding must not be too strong or weak
• Ideal KD should be between 10-4 & 10-8 M
• Elution involves passage of high salt or low
pH buffer after binding
Ligand
Specificity
AMP
Enzymes with NAD cofactors an ATP
dependent kinases
Arginine
Proteases such as prothrombin,
kallikrein, clostripain
Cibacron Blue Serum Albumin, Preablumin
Dye
Heparin
Protein A
Calmodulin
EGTA-copper
Growth factors, cytokines, coagulation
factors
Fc region of immunoglobulins
Calmodulin regulated kinases, cylcases
and phosphatases
Proteins with poly-Histidine tails
Size Exclusion Chromatography (SEC)
• Molecules are separated according to
differences in their size as they pass
through a hydrophilic polymer
• Polymer beads composed of cross-linked
dextran (dextrose) which is highly porous
(like Swiss cheese)
• Large proteins come out first (can’t fit in
pores), small proteins come out last (get
stuck in the pores)
SEC
Sephadex Structure
Ion Exchange Chromatography
(IEC)
• Principle is to separate on basis of
charge “adsorption”
• Positively charged proteins are
reversibly adsorbed to immobilized
negatively charged beads/polymers
• Negatively charged proteins are
reversibly adsorbed to immobilized
positively charged beads/polymers
IEC
• Has highest resolving power
• Has highest loading capacity
• Widespread applicability (almost
universal)
• Most frequent chromatographic
technique for protein purification
• Used in ~75% of all purifications
IEC Principles
IEC Nomenclature
• Matrix is made of porous polymers derivatized
with charged chemicals
• Diethylaminoethyl (DEAE) or Quaternary
aminoethyl (QAE) resins are called anion
exchangers because they attract negatively
charged proteins
• Carboxymethyl (CM) or Sulphopropyl (SP)
resins are called cation exchangers because
they attract positively charged proteins
IEC Groups
IEC Techniques
• Strong ion exchangers (like SP and QAE)
are ionized over a wide pH range
• Weak ion exhangers (like DEAE or CM) are
useful over a limited pH range
• Choice of resin/matrix depends on:
–
–
–
–
Scale of separation
Molecular size of components
Isoelectric point of desired protein
pH stability of the protein of interest
Protein pH Stability Curve
+
Net charge on protein
Attached to
anion exchangers
4
5
6
7
8
Attached to
cation exchangers
_
Range of pH stability
9 pH
Polyacrylamide gel electrophoresis and N-terminal amino acid
sequence of D-carnitine dehydrogenase
1. Polyacrylamide gel electrophoresis
A. Native-PAGE
B. SDS-PAGE
1.
2.
3.
4.
Coomasive brilliant blue R-250 staining
Activity staining
Marker proteins
Purified enzyme
2. N-terminal amino acid sequence of D-carnitine dehydrogenase
5
10
15
20
25
30
M Q N LR R V LI TAAX S G I G R E IAKAF V N E G H L
Estimation of the molecular weight of D-carnitine
dehydrogenase
1. Membrane
(Module)
2. Pumps
3. Other
•Piping
•Tanks
•Valves
•Flowmeter
•Manometer
4/9/2015
FEATURE
REVERSE
OSMOSIS
NANO
FILTRATION
ULTRA
FILTRATION
Membrane
Asymmetrical
Asymmetrical
Asymmetrical
Wall Thickness
150mm
150mm
150-250mm
10-150mm
Film thickness
1mm
1mm
1mm
various
Pore size
<0.002um
<0.002um
0.02-0.2um
0.2-5um
HMWC, mono,di-, and aligosaccharides,
polyvalent
anions
Macromolecules,
proteins,
polysaccharid
es, viruses
Particulates, clay,
bacteria
Tubular, spiralwound, plate
& frame
Tubular, hollowfibre, spiralwound, plate
& frame
Tubular, hollowfibre, plate &
frame
CA, TFC,
Ceramic,
PVDF,
Sintered
Rejects
Membrane
module
HMWC,
LMWC,Sodiu
m, Chloride,
glucose,
amino acids,
proteins
Tubular, spiralwound, plate
& frame
MICRO
FILTRATION
Symmetrical
Asymmetrical
Material
CA, TFC
CA, TFC
CA, TFC, Ceramic
Pressure
15-150 bar
5-35 bar
1-10 bar
<2 bar
Flux
10-50 (l/m2/hr)
10-100 l/m2/hr
10-200 l/m2/hr
50-1000 l/m2/hr
Some membrane types
Ultrafiltration
RO membrane
Microfiltration
MIKROFILTRASI
Tanpa pembentukan cake
•Menggunakan membran: tipis dan
microporous
•Lubang pori2nya kecil dan sangat
monodisperse
•Mempunyai kemampuan menyaring partikel
yang tidak diinginkan
•Membran mengikuti hukum Darcy’s untuk
permeabilitas dan ketahanan yang tinggi thd
aliran. Konvensional ketahanannya rendah.
•Perlu dilakukan pembersihan secara berkala
•Aliran yang melalui membran lebih rendah
daripada aliran melalui conventional filter
cake. Filter area per liter volume lebih besar
daripada convensional.
•Type : Plate and frame, spiral wound and
hollow fiber
Plate and Frame
Spiral Wound
Tubular ceramic
Hollow Fibre
Bacterial Cell
Casein, whey
Lactose
Minerals
Important terms
• Feed or Product
– Initial material into system on
feed side of membrane
• Retentate or Concentrate
– The fraction of the feed which
is rejected by the membrane.
• Permeate
– The fraction of the feed which
passes through the membrane
• Spiral Wound
• Plate and Frame
• Tubular
• Capilary
• Hollow fibre
• Pipe
• Ceramic
• Zeolite
• Stainless Steel
Downstream protein purification
by ultrafiltration concentration and
diafiltration
MF Applications: late 1990s
•
•
•
•
•
•
•
•
•
•
Cold sterilization of pharmaceuticals
Cell harvesting
Sterile process filters for gas-phase
Clarification of fruit juices, wine and beer
Ultrapure water in semiconductor industry
Metal recovery (colloidal (hydro)oxides)
Waste water treatment
Separation of oil-water emulsions
Dehydration of lattices
Pretreatment for RO
Eykamp, 1995; Mulder, 1998
UF Applications: 1980s
• Chemical Industry
•Electro coat painting recovery
•Latex processing
•Textile size recovery
•Recovery of lubricant oils
• Medical Applications
•Kidney dialysis
• Waste treatment
• Recovery of valuable products from
effluents
•Cheese whey
Cheryan, 1986
UF Applications: late 1990s
•
•
•
•
•
•
electro paint recovery, oil-water emulsions
Beverages (juices)
Dairy (milk, whey, cheese making)
Food (gelatin, starch, sugar and proteins)
Textile (sizing, dyes)
Pharmaceutical (enzymes, antibiotics,
pyrogens)
• Pulp and paper industry
• Leather industry
• Water purification
Eykamp, 1995; Mulder, 1998
Factors affecting membrane
structure:
• choice of polymer, choice of solvent and
nonsolvent, composition of casting solution,
composition of coagulation bath, temperature
of the casting solution and coagulation bath,
evaporation time, location of the liquid-liquid
demixing gap and crystallization behaviour of
the polymer
Module Type
Characteristic
Flat plate
Spiral
Wound
Shell
and
Tube
Hollow Fibre
Packaging
density (m2/m3)
Moderate
(200-400)
Moderate
(300-900)
Low
High (9000-30000
(150-300)
Fluid
management
Good
Good
High
pumping
costs
Good
Suspended
solids
capability
Moderate
Poor
Good
Poor
Cleaning
Sometimes
difficult
Sometimes
difficult
Easy
Backflusing possible
Replacement
Sheets or
cartridge
Cartridge
Tubes
Cartridge
DRYING
Reason: The cost of transport can be reduced; the
material is easier to be handle and package; can be
more conveniently stored in the dry state; more
stable than the liquid form.
Instrument: spray dry, in this system the evaporative
cooling protect the enzyme activity.
CRYSTALLIZATION
Is the best way to preserve the enzyme, but the
method for most enzyme still to be developed. The
enzyme should be pure.
Why immobilized enzymes?
Definition : Immobilization means that the biocatalysts are limited in moving
due to chemically or physically treatment
Reasons
Limitations
- Reuse of enzyme(reducing cost)
-Cost of carriers and immobilization
- Easy product separation
-Changes in properties(selectivity)
- Continuous processing
-Mass transfer limitations
- Stabilization by immobilization
-Activity loss during immobilization
Conventional Immobilization Methods
Adsorption
Covalent
binding
Cross-linking
Immobilization
Entrapment
Encapsulation
Immobilization
type
Adsorption /
Absorption
Generally simple
Advantage
Disadvantage
 No manipulation of the
protein sample
 Some protein denature
and inactive
 Unstable binding Nonspecific random and
multi-oriented protein
immobilization: activity
decreased
 Irreproducibility of
results
Covalent crosslinking
 Reproducibility and
stability of protein layer
 The possibility of
controlling the density
and environment of the
immobilized species
 Non-specific random
orientation: activity
decreased
 Some protein denature
and inactive
 Additional chemical
reaction for modification
in vitro
Affinity attachment
 Identical orientation by
site specific
immobilization
 Direct immobilization
by high affinity
 Easy protein
purification and array
fabrication
 Reproducibility and
stability of protein layer
 The possibility of
controlling the density
and environment of the
immobilized species
 Difficult application in
multi-subunit proteins
 The possibility of
elusion of some protein