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

Albumin
Ovalbumin
Carbonic anhydrase
Trypsin inhibitor
-lactalbumin

Mr (kDa)
Source
97
Rabbit muscle

66
45
30
20,1
14,4

Bovine serum
Chicken egg white
Bovine erythocyte
Soybean
Bovine Milk

Amount (g)
67

83
147
83
80
116

HMW-SDS Marker
Protein
Myosin
-2-Macroglobulin
-Galactosidase

Transferrin
Glutamate
dehydrogenase

Mr (kDa)
Source
220
Rabbit muscle
170
Bovine plasma
116
Escherchia coli

76
53

Human
Bovine liver

Amount (g)
25
100
16

17
18

HMW-Native Marker
Protein
Thyroglobulin

Ferritin
Catalase
Lactate dehydrogenase
Albumin

Mr (kDa)
Source
669
Porcine thyroide

440
232
140
66

Equine spleen
Bovine liver
Bovine heart
Bovine serum

Amount (g)
76

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

Prosedur Kerja

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

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

6 7 8

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