ELECTROPHORESIS

FOOD ANALYSIS AND BIOCHEMISTRYPRACTICE

ENDRIKA WIDYASTUTI MOCH. NURCHOLIS
FOOD AND SCIENCE TECHNOLOGY UNIVERSITY OF BRAWIJAYA 2012

Electrophoresis
Separation into bands due to friction through the gel and charge on protein. Magnitude of charge and voltage will also determine how far the protein will travel in the electrical field. Smaller proteins tend to move faster

Why Electrophoresis ???

Quantiative analysis and fractination of biological fluids Characterization of purified components Detection and characterization of macromolecular interactions

Type of Electrophoresis
Moving boundary electrophoresis Zone electrophoresis SDS Disk Electrophoresis Paper electrophoresis SDS-PAGE

Electrophoresis- SDS Page

Separation based on size.
Protein berikatan dengan SDS to become negatively charged. SDS = sodium dodecyl sulfate => anionic detergent (negative charge) Proteins move through gel matrix to the anode (electrical pole with a positive charge). The RATE they move is based on size. Good for determining protein composition, purity, and estimation of molecular weight.

ELEKTROFORESIS SDS-PAGE
Prinsip Analisis : Suatu metode untuk memisahkan makromolekul seperti asam nukleat dan protein berdasarkan ukuran, muatan listrik dan ciri fisik.
Tujuan : Mengetahui prinsip dasar pemisahan dengan metode elektroforesis Menentukan berat molekul kasein

protein

ELEKTROFORESIS SDS-PAGE
Protein mempunyai muatan positif dan negatif Muatan listrik menyebabkan protein bergerak ke elektroda melewati gel poliakrilamid Gel memisahkan molekul berdasarkan : 1. Ukuran 2. Bentuk molekul 3. Kekuatan medan listrik 4. Sifat hidrofobik relatif sampel 5. Kekuatan ionik.
Poliakrilamid memisahkan Protein MW 0,5-250 kDa memisahkan DNA 5-2000 bp

Isoelectric Point (pI)

Setiap protein memiliki (pI), kondisi dimana protein tidak bermuatan sehingga tidak terjadi perpindahan.
pH dimana protein tidak bermuatan Protein dikatakan basa, asam atau netral tergantung pada muatan protein pada pH fisiologis

Nilai pH dibawah pI protein berpindah sebagai kation(-) mobility increasing with decreasing pH Nilai pH diatas pI protein berpindah sebagai anion(+), mobility increasing with increasing pH

Media for Electrophoresis

Paper strip Cellulose acetate Agar Starch Polyacrylamide gels (PAGE) molecular sieving is utilized to great advantage. PAGE Size, shape and electrophoretic mobility, Improved resolution

MARKER
LMW (Low Molecular Weight) 14,4-97kDa HMW-SDS (High Molecular Weight) 53-220 kDa HMW-Native 66-669 kDa Peptide marker kit (Horse myoglobin peptides) Mr = 2,5-17 kDa

LMW Marker
Protein Phosphorylase b Mr (kDa) Source 97 Rabbit muscle Amount (g) 67

Albumin Ovalbumin Carbonic anhydrase Trypsin inhibitor -lactalbumin

66 45 30 20,1 14,4

Bovine serum Chicken egg white Bovine erythocyte Soybean Bovine Milk

83 147 83 80 116

HMW-SDS Marker
Protein Myosin -2-Macroglobulin -Galactosidase Mr (kDa) Source 220 Rabbit muscle 170 Bovine plasma 116 Escherchia coli Amount (g) 25 100 16

Transferrin Glutamate dehydrogenase

76 53

Human Bovine liver

17 18

HMW-Native Marker
Protein Thyroglobulin Mr (kDa) Source 669 Porcine thyroide Amount (g) 76

Ferritin Catalase Lactate dehydrogenase Albumin

440 232 140 66

Equine spleen Bovine liver Bovine heart Bovine serum

50 36 48 40

Bahan-Bahan

Sampel Kasein Buffer Bufer Tris-Cl 0,5 M pH 6,8 ; SDS 2% ; Merkaptoetanol 0,05% Larutan stock Akrilamid 30% 29.2 gram akrilamid ditambah 0.8 gram NN-bis-methylene acrylamid dalam 100 ml aquades.

Bahan-Bahan
Larutan SDS 10 % Amonium persulfat (APS) 10% (di buat setiap akan digunakan) TEMED Larutan Pewarna (Staining) 0.1 % commasie blue dalam larutan metanol : air : asam asetat (5:5:2) Larutan Pembilas (destaining) metanol : air : asam asetat (5:5:2) Aquades

Alat
Seperangkat alat elektroforesis Mikropipet Tip Beker glass 100 ml Beker glas 50 ml Eppendorf Shaker

Seperangkat Alat Elektroforesis

Prosedur Kerja
Pembuatan gel : Pasanglah alat gelas untuk mencetak gel ke tempat yang disediakan (seperti gambar 4.) Untuk membuat 20 ml gel 20% campurkan 13.3 ml larutan stok akrilamid 30%, 5 ml buffer Tris-HCl 0.5 M, pH 6.8, 0.2 ml SDS 10%, 1.5 ml aquades. Tambahkan segera 100 l APS 10% dan TEMED 10 l Aduk hingga tercampur merata Tuangkan ke dalam cetakan gel dengan menggunakan mikropipet hingga tinggi yang dikehendaki. Beri sisa tempat untuk stacking gel di bawah area peletakan gigi sisir. Biarkan gel terpolimerisasi selama 15-30 menit dalam suhu ruang.

Polyacrylamide Gel
Cathode

Anode

Proteins separated by molecular weight

Prosedur Kerja
Tuangkan aquades dengan mikropipet ke permukaan gel pemisah dan kemudian buang aquades tersebut dengan menyerapkan tisu Sementara itu buat lagi gel untuk membuat 4 ml stacking gel 4% dengan mencampur 1.2 ml larutan stok akrilamid 30%, 0.5 ml buffer Tris-HCl 6.8, 40 l SDS 20%, 2.26 ml aquades. Tambahkan segera 20 l 10% APS dan 5 l TEMED. Tuangkan larutan ke atas gel pemisah Sisipkan gigi sisir pada stacking gel dengan perlahan, jangan sampai terbentuk gelembung. Biarkan gel terpolimerisasi selama 15-30 menit dalam suhu ruang Ambillah sisir secara perlahan dari gel. Pindahkan gel secara perlahan ke dalam tank elektroforesis (seperti gambar 5.) Masukkan buffer tank ke dalam tank elektroforesis

 Persiapan sampel Larutkan 0.1 gram kasein ke dalam 4.9 gram sampel buffer Panaskan pada suhu 90oC selama 5 menit.

Prosedur Kerja

Pemisahan protein dengan elektroforesis Masukkan sampel ke dalam sumuran sebanyak 5 l Pasanglah elektrode sesuai dengan warnanya. Gel dijalankan pada tegangan 200 V selama 45 menit atau hingga sampel telah mencapai bagian dasar.

Pemisahan Molekul Berdasarkan Berat Molekul dan Muatan

Proses Elektroforesis

Pewarnaan Gel
Hentikan listrik, pindahkan gel dari tank Pindahkan glass plate dari gel kedua sisi Tuangkan larutan pewarna pada gel dalam wadah Tutup dengan plastik dan letakkan di atas shaker selama 15-30 menit Pindahkan larutan pewarna dari gel. Simpan untuk digunakan kembali. Bilas gel dengan aquades Tuangkan larutan pembilas selama dan masukkan potongan kertas saring, biarkan selama 10-15 menit di atas shaker Ganti larutan pembilas dengan yang baru hingga yang terlihat pada gel adalah pita-pita protein.

Pengamatan

Amati pita-pita yang terbentuk pada gel elektroforesis. Cari dalam literatur berat molekul masing-masing komponen penyusun kasein dan tentukan letak komponen tersebut pada pita gel elektroforesis.

Hasil SDS-PAGE
1 2 3 4 5 6 7 8 9 M

Protein gel (SDS-PAGE) that has been stained with Coomassie Blue.

MATERI TAMBAHAN

SDS-PAGE (PolyAcrylamide Gel Electrophoresis)

SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, is a technique widely used in biochemistry, forensics, genetics and molecular biology: to separate proteins according to their electrophoretic mobility (a function of length of polypeptide chain or molecular weight). to separate proteins according to their size, and no other physical feature.

Fig.1Before SDS: Protein (pink line) incubated with the denaturing detergent SDS showing negative and positive charges due to the charged R-groups in the protein. The large H's represent hydrophobic domains where nonpolar R-groups have collected in an attempt to get away from the polar water that surrounds the protein. After SDS: SDS disrupt hydrophobic areas (H's) and coat proteins with many negative charges which overwhelms any positive charges the protein had due to positively charged R-groups. The resulting protein has been denatured by SDS (reduced to its primary structure-aminoacid sequence) and as a result has been linearized.

..SDS
SDS (the detergent soap) breaks up hydrophobic areas and coats proteins with negative charges thus overwhelming positive charges in the protein. The detergent binds to hydrophobic regions in a constant ratio of about 1.4 g of SDS per gram of protein.

..SDS
Therefore, if a cell is incubated with SDS, the membranes will be dissolved, all the proteins will be solubalized by the detergent and all the proteins will be covered with many negative charges.

PAGE

If the proteins are denatured and put into an electric field (only), they will all move towards the positive pole at the same rate, with no separation by size. However, if the proteins are put into an environment that will allow different sized proteins to move at different rates. The environment is polyacrylamide. the entire process is called polyacrylamide gel electrophoresis (PAGE).

..PAGE
Small molecules move through the polyacrylamide forest faster than big molecules. Big molecules stays near the well.

The actual bands are equal in size, but the proteins within each band are of different sizes.

Sample of SDS- PAGE