METODE SDS- PAGE

Oleh:
Susila Kristianingrum
Langkah SDS-PAGE

1.Pembuatan gel pemisah (separating gel)

2. Pembuatan gel pengumpul (stacking gel /
upper gel)
3. Pemanasan sampel
4. Dilarutkan. Runing pada plate 1 dengan
arus 28A dengan tegangan 110V dan pada
plate 2 dengan arus 30A dan tegangan 130 V.
5. Gel direndam dalam larutan staining
untuk proses pewarnaan selama 20 menit
6. Gel direndam dalam larutan destaining
(Penghilangan warna) untuk proses
pencucian 20 menit
 Elektroforesis SDS-PAGE
• Pemisahan protein
(molekul bermuatan
berdasarkan ukuran dalam
medan listrik
• Protein didenaturasi dengan
detergen anion
Analisis Tingkat Kemurnian dengan
SDS-PAGE

1. Ekstrak kasar
2. Pengendapan dengan garam
3. Kromatografi penukar ion
4. Kromatografi filtrasi gel
5. Kromatografi afinitas

10μg protein/lajur
Penentuan Massa Molekul Protein
 SDS-PAGE
(Sodium Dodecyl Sulphate-
Polyacrylamide Gel
Electrophoresis)

Working with Proteins: Protein Isolation and

Characterization
(Fig 1-8)
 [2] Solubilization
1. Osmoticlysis: simple and gentle
- place cells in a hypotonic solution
2. Mechanical: good if cells have cell wall
crushing or grinding of cells,
French press (pressure), sonication (sound)
3. Detergents: used if protein is in lipid membrane

• pH: use an appropriate buffer. Figure 2-16; p.13, H(3)

• Temperature: 0-4 °C – can be unstable once outside of cell
• Proteases: must be inhibited (Proteases (proteinases) cut
amide bonds of protein)
• Adsorption: keep concentrated (denatures at surfaces)
minimize foaming/exposure to air (reduce unfolding)
• Storage: store under inert gas, frozen at -80 °C or -196 °C
[3] Stabilization
Proteins are most stable under pH and ionic strength close to
physiological conditions.
pH: 7.4, (lysosomal enzymes, pH 5)
I (ionic strength) = 0.15 M

 How is Ionic Strength determined?

I = ½ciZi2
where ci is the concentration (mol/L) of ionic species,
and Zi is the net charge of the species.

① 0.1 M sodium acetate, CH3COONa

I = ½ ([Na+]12 + [CH3COO-]12) = ½ (0.11 + 0.11)
= 0.1 M (equal to molarity)
② 0.1 M Na2HPO4
I = ½ ([Na+]12 + [HPO42-]22) = ½ (0.21 + 0.14)
= 0.3 M (larger than molarity)
Solubility of carboxy-hemoglobin at its isoelectric point
as a function of ionic strength and ion type.

NaCl

KCl
S
log
S’ MgSO4
Na2SO4

K4SO4

Ionic Strength
 [4] Solubilities of Proteins

Salting In
Addition of salt at low ionic strength can increase
solubility of a protein by neutralizing charges on the surface
of the protein, reducing the ordered water around the
protein and increasing entropy of the system.

Salting out (Can be used for Fractionation)

If the concentration of neutral salts is at a high level
(>0.1M), in many instances the protein precipitates.
This phenomenon apparently results because the
excess ions (not bound to the protein) compete with
proteins for the solvent. The decrease in solvation and
neturalization of the repulsive forces allows the proteins to
aggregate and precipitate. This effect is called "salting
out".
The effect of salt on different proteins may differ:

Certain proteins precipitate from solution under conditions

In which others remain quite soluble.

Once the protein is precipitated (not denatured) –

can separate by centrifugation  pellet
can be redissolved in buffer for further purification

Ⓠ Which protein will precipitated first? (hydrophobic or

hydrophilic?)
 [5] Dialysis
• Following a salting-out step, the solution will contain a high
concentration of salt that can be disruptive to subsequent
chromatographic steps.
• The salt can be removed by dialysis – dialysis tubing has pores
with a specific molecular weight cut-off that allows smaller
molecules (salt) to pass.

Buffer– large volume

Dialysis tubing with protein and high salt

Exchange buffer
> 3 times
[6] Separation - Chromatography
Makes use of a mobile phase (fluid - usually buffer)
& a stationary phase (usually small beads).
Ion exchange chromatography
 pH and [salt] dependent
 Separates by ionic charge:
cations & anions

Cation exchange
Anion exchange
Where on the pH scale should the buffer
be compared to the pI of the protein?

This is an example of cation exchange.

How will anion exchange work?

Cation exchanger:
CM-cellulose -CH2-COO-

Anion exchanger:
C2H5
DEAE-cellulose -CH2-CH2-NH-C2H5
+
Choosing an ion exchanger will depend on:

The pI value of the target protein.

The pH of buffer used.

An (acidic) protein of a pI of 5.

At pH 7 (phosphate buffer or Tris buffer),

the protein will carry a net negative charge.
 The protein will bind to an anion exchanger (DEAE)
 Will be repelled from a cation exchanger (CM).

• Apply the protein mixture containing the target protein.

• Remove neutral and basic proteins in flow-through.
• Recover the target protein with increasing [salt].
Size exclusion chromatography
Gel filtration chromatography

 Contains porous beads

 Separates according to size and shape
 Larger proteins excluded
from the small pores
 Quaternary structure determination,
& Mr estimation using
a standard curve
(log Mr vs elution volume)

Ⓠ Fibrous proteins
Spherical vs rod-shaped proteins
Affinity chromatography
 Separates by specific interactions
 Contains a ligand:
enzyme ‒ substrate
receptor ‒ hormone
antigen ‒ antibody
(His)6 protein – Ni2+
Electrophoretic Analysis
Separates according to size and charge concentration
Matrix is polyacrylamide (proteins) or agarose (nucleic acids)

SDS-PAGE (polyacrylamide gel electrophoresis):

uses sodium-dodecyl sulphate (SDS) to coat the proteins
to give them all equivalent (negative) charge concentration
– proteins can then separate by M.W. only
SDS-PAGE

s - soluble fraction
i - insoluble fraction
p - post-Ni2+ column
SDS-PAGE Animation
Isoelectic focusing

 Ampholytes are low M.W. organic acids and bases

that distribute along the electric field across the gel.
 Each protein of a mixture distributes across the gel
according to their pI.
Isoelctric focusing can be described as electrophoresis in a pH gradient set
up between a cathode and anode with the cathode at a higher pH than the
anode. Because of the amino acids in proteins, they have amphoteric
propertites and will be positively charged at pH values below their pI and
negatively charged above. This means that proteins will migrate toward their
pI. Most proteins have a pI in the range of 5 to 8.5.
Under the influence of the electrical force the pH gradient will be established
by the carrier ampholytes, and the protein species migrate and focus
(concentrate) at their isoelectric points. The focusing effect of the electrical
force is counteracted by diffusion which is directly proportional to the protein
concentration gradient in the zone. Eventually, a steady state is established
where the electrokinetic transport of protein into the zone is exactly balanced
by the diffusion out of the zone.
 Isoelectric focusing
carrier ampholite; oligoamino, ologicarboxylic acids of 600~900 Da
R-NH-(CH2)n -N-(CH2)n-COOH
R=H, -CH2COOH, CH2-N-R2R2
2-D electrophoresis

Perhaps certain proteins have

identical pIs or molecular weights

1. Separate by IEF – 1st dimension

2. Separate by SDS-PAGE – 2nd dimension
this gives a second dimension to the analysis
Sumber:
David L. Nelson and Michael M. Cox. 2004. Lehninger Principles of
Biochemistry. Fourth Edition. akses 11 November 2012.
W. H. Freeman & Company.

Uregina.ca/suhdaey/courses/.../08%20H5.ppt akses 11 November

2012.