Native PAGE Klmpk 1

NATIVE PAGEELECTHROPORESIS UNDER NON DENATURING CONDITION

ANGGOTA KELOMPOK

Faizal Agung Pratomo 0911310011Putri Akte Susanti 0911310023Deshinta Rizky P 0911310037Prima Santi 0911310056Tri 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 lainnya3. Oligomer atau agregat4. Binding event (protein – protein atau protein ligan)

1. Blue Native (BN-PAGE)2. Clear Native (CN-PAGE)3. Quantitative Preparative Native

Continuous (QPNC-PAGE)

Methods are . . .

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 dari2 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.