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NATIVE PAGE

ELECTHROPORESIS UNDER NON DENATURING


CONDITION
ANGGOTA KELOMPOK
Faizal Agung Pratomo 0911310011
Putri Akte Susanti 0911310023
Deshinta Rizky P 0911310037
Prima Santi 0911310056
Tri Widyanti 0911310067
Ling Sandra A.H.A 0911313027
Native Page is . . .

Native Polyacrylamide Gel


Electrophoresis merupakan metode
pemisahan elektroforesis khas yang
menggunakan proteomics dan
metallomics pada kondisi non-
denaturing.
Native Polyacrylamide Gel
Electrophoresis untuk protein
fungsional, isoenzyme, isoelektrik
Untuk mengetahui enzim dengan
fungsi yang sama
Aims are....
 Untuk mendeteksi :
1. Perubahan muatan akibat degradasi kimia
(misalnya deaminasi)
2. Unfolded atau peristiwa perubahan
konformasi lainnya
3. Oligomer atau agregat
4. Binding event (protein – protein atau protein
ligan)
Methods are . . .
1. Blue Native (BN-PAGE)
2. Clear Native (CN-PAGE)
3. Quantitative Preparative Native
Continuous (QPNC-PAGE)
Blue Native (BN-PAGE)
Memisahkan kompleks protein dengan menggunakan
pewarna Coomassie Brilliant Blue.
Kerugian :
 Pewarna berikatan dengan komplek protein

sehingga komplek protein memisah


 Memiliki potensi quenching chemoluminescence

(berpendar)
Clear Native (CN-PAGE)
Memisahkan asam larut air dan protein membran
menggunakan gradien gel poliakrilamin.
Pemisahan berdasarkan muatan intrinsik protein
Keuntungan : Tidak menunggukan pewarna, dapat
menahan kompleks protein membran supramolekul
Quantitative Preparative Native Continuous (QPNC-
PAGE)

Pemisahan komplek protein yang didenaturasi


dengan buffer elektroforesis (pH 10), dilanjutkan
dengan cairan fisiologis (pH 8)
Protocol.....
Example Native Page
 Alat :
 Bio-Rad Criterion Precast Gel System
 Model # CRITERION Cell
 Thermo Electron 2060P Power Supply
 Belly Dancer Shaker
 Bio-Rad GelAir Dryer
 Pipetman P20 Micropipet, 2-20 L
 Pipetman P200 Micropipet, 20-200 L
 Pipetman P1000 Micropipet, 100-1000 L
 Beckman Microfuge 11
 Gel Cutter Acrylic, Sigma (Cat. # G4778
Sampel
 PEC or PEC-HL samples
 Purchased amylase, carboxypeptidase, elastase, lipase
and proteases, trypsin and chymotrypsin.
Material
 Bio-Rad Criterion Precast Polyacrylamide Gels (8.7 x 13.3 cm; 1.0 mm
thick); 18 well; 30 L well capacity
 Native (Cat. # 345-0033): 4-20% acrylamide (Tris-HCl), 2.6% bis-acrylamide
crosslinker
 SDS (Cat. # 345-0033): 4-20% acrylamide (Tris-HCl), 2.6% bis-acrylamide
crosslinker
 IEF (Cat. # 345-0072): pH 3-10, 5% acrylamide, 3.3% bis-acrylamide crosslinker
 10X Tris/Glycine Running Buffer, [concentration of 1X is 25 mM Tris, 192
mM glycine, pH 8.3]
 10X Tris/Glycine/SDS Running Buffer, [concentration of 1X is 25 mM Tris,
192 mM glycine, 0.01% SDS, pH 8.3]
 Native Sample Buffer, [62.5 mM Tris-HCl, pH 6.8, 40% glycerol, 0.01% w/v
bromophenol blue]
 Precision Plus Protein Standards – All Blue, Bio-Rad (Cat. # 161-0373)
 Laemmli Sample Buffer, Bio-Rad (Cat. # 161-0737) [62.5 mM Tris-HCl, pH 6.8,
2% SDS, 25% glycerol, 0.01% w/v bromophenol blue]
 -mercaptoethanol, electrophoresis grade, Sigma (Cat. # M7154)
 Imperial Protein Stain, Pierce (Cat. # 24615)
 IEF Standards, Broad Range pI 4.45-9.6, Bio-Rad (Cat. # 161-0310)
 10X IEF Cathode Buffer, Bio-Rad (Cat. # 161-0762) [concentration of 1X is 20 mM
lysine, 20 mM arginine]
 10X Anode Buffer, Bio-Rad (Cat. # 161-0761) [concentration of 1X is 7 mM
phosphoric acid]
 IEF Sample Buffer, [50% glycerol]
 FisherBrand Sterile Gel Loading Tips, 1-200 L (Cat. # 02-707-81)
 Nalgene Round Floating Microcentrifuge Tube Rack
 Gel Drying Solution: 1X, Bio-Rad (Cat. # 161-0752) [contains water, ethanol]
 GelAir Cellophane Support, Bio-Rad (Cat. # 165-1779)
1. Preparation of 1X Running
Buffer
• Add 100 mL 10X Tris/Glycine Running Buffer to 900
mL distilled water. Mix thoroughly.
2. Preparation of Samples
• Accurately weigh out 20-25 mg of pancreatin enzyme
concentrate (PEC) or pancreatin enzyme concentrate
– high lipase (PEC-HL) and transfer to a 2.0 mL
microcentrifuge tube. Add 1.0 mL distilled water and
vortex vigorously for five minutes. Centrifuge samples
at 2000 x g (8000 rpm on Beckman Microfuge 11) for
ten minutes. Dilute 50 L supernatant with 100 L
Native Sample Buffer.
3. Preparation of Criterion Precast Gel

• Remove precast gel from storage container and rinse


with a few squirts of distilled water. Place cassette in
one of the slots in the Criterion tank. Add
approximately 40 mL 1X Tris/Glycine Running Buffer
to upper chamber. Gently remove well comb by pulling
upward in a uniform motion.
4. Loading of Samples
• Using a micropipet with gel-loading tips, load 20 L of
each sample per well.
5. Running Conditions
• Once samples have been loaded, add approximately 400
mL 1X Tris/Glycine Running Buffer to the lower
chamber of the cell (up to the FILL line). Snap the lid on
the chamber and plug the leads into the power source.
Place the chamber in the cold room. Apply a constant
voltage of 125 V for 120 minutes; monitor and record the
initial and final currents.
6. Staining Protocol
• After electrophoresis is complete, turn off the power
supply and disconnect the electrical leads. Remove the
Criterion cassette and pour off the buffer from the
upper chamber. Open the cassette by inverting it and
cracking the welds using the tool built into the lid of the
tank. Transfer the gel to a Nalgene staining container.
Wash gel once for five min using 200 mL distilled water.
Shake on an orbital shaker at 55 rpm throughout wash.
Remove all water from the staining container.
Continue..
 Add approximately 100 mL Imperial Protein
Stain (enough to completely cover gel) and shake
at 55 rpm for one hour. Rinse gel in 200 mL of
distilled water while shaking at 55 rpm. Place a
folded KimWipe in the staining container during
the destain step to absorb excess stain and decrease
the time needed to fully destain the gel. After
destaining, dry gel according to gel drying
protocol that follows the separation protocols in
this document.
Drying of Acrylamide Gels
 Gel Preparation
 Frame Assembly
 Drying Conditions
factors
Preparasi Sampel
 Sebaiknya ditambahkan inhibitor protease ke dalam
sampel untuk menjaga aktivitas biologis dari
sampel protein
Sentrifugasi
 Setiap material harus larut untuk
mencegah goresan pola dalam gel
sehingga dilakukan sentrifugasi.
Salt
 Pengaruh salt terhadap native page dengan media
gradien PhastGel akan tergantung pada konsentrasi
garam, jenis garam, dan pH sampel.
 Konsentrasi garam tinggi (1 M) di kombinasi
dengan pH rendah (pH 4) akan mempengaruhi hasil
yang buruk
Konsentrasi Sampel
 Sensitivitas teknik pengembangan dan volume
sampel yang dilakukan pada gel akan menentukan
batas bawah konsentrasi sampel.
 Sampel yang mengandung lebih dari
2 mg / ßl (Coomassie) atau 100 ng / ßl (perak) dari
masing-masing protein dapat berakibat kelebihan
gel dan mendistorsi hasilnya.
Examples
Native Page. .
 For acidic proteins, Laemmli's gel
system without SDS can be used for
native PAGE. An example is shown
above in Fig.1. The glycoprotein
used here, recombinant human
erythropoietin (EPO, lane 1) is
highly sialylated and hence
negatively charged at the pH of
Laemmli's system, pH=8.4. Fig.1
also shows the results of native
PAGE for the protein after being
stressed by heating at 79 °C. As the
incubation time is increased (lanes 2-
5) there is increasing formation of a
new band, corresponding to dimers,
as confirmed by both sedimentation
velocity and non-reducing SDS
PAGE analysis.
 It is therefore possible to
screen conditions that
minimize such oligomer
formation using native
PAGE. In fact, the figure
above shows no dimer
formation in histidine,
glycine or Tris-HCl buffers
(all at 20 mM), consistent
with the highly-reversible
thermal unfolding of EPO
in those conditions.
 example of native PAGE for an acidic protein,
BSA, obtained from 2 different sources. In this
case, the gel was run at 60 °C to examine the
actual events occurring during unfolding of BSA
at 60 °C. Many bands are observed for BSA after
heating in buffer alone (lanes 1 and 4, from
different suppliers). Fewer bands are observed
when the BSA sample contains detergents (i.e., it
is protected by the detergents), as in lanes 2-3
and 5-7. For basic proteins, acid-urea gels or
acid gels are often used. However, most proteins
denature to some extent at acidic pH and in the
presence of urea. Under such denaturing
conditions, the mobility of proteins may not
reflect their conformation at physiological pH. In
addition, protein-protein interactions or
aggregation of proteins that occurs at normal pH
may be altered. Therefore, we run native gels for
basic proteins under more physiological
conditions, i.e., pH 6.1 and in the absence of
urea. 
 an example of a protein that has an
isoelectric point (pI) above 8. Lanes
1-6 correspond to the same protein
processed or stored differently. Only
one sample shows an extra band
with higher mobility, reflecting
some form of chemical degradation
under this particular condition.
Therefore, one can use native-PAGE
to screen conditions that eliminate
such degradation. Since the mobility
of the degradation product is greater,
it should have more positive charges
than the starting protein at the pH of
the gel, pH=6.1.
 This native PAGE system for basic
proteins can detect aggregation as with
Laemmli's system. As an example, the
same protein used in Fig. 4 was heated
in 5 different buffers and examined for
the degree of aggregation. As shown
above in Fig. 5, the band corresponding
to the monomer (highest mobility)
decreases and new bands corresponding
to aggregates appear upon heating,
while only one band is observed in these
buffers before heating (as in lanes 1-3 in
Fig. 4). The degree of aggregation is
highly dependent on the type of buffer.
In one condition (lane 5), almost no
aggregation occurred, indicating that
under this condition aggregation of this
protein is greatly reduced.