KROMATOGRAFI

AFINITAS
Fitria Susilowati S.Pd., M.Sc.
Universitas Darussalam Gontor
 PENDAHULUAN
Pemurnian enzim atau protein menggunakan
teknik kromatografi afinitas pada saat ini
sangat populer dan menjadi pilihan utama.
Pemurnian ini dilakukan berdasarkan afinitas
enzim atau protein terhadap biomolekul lain
(ligan), misalnya enzim terhadap inhibitor,
substrat atau produknya, afinitas antibodi
terhadap antigennya, atau afinitas hormon
terhadap reseptornya.
 Afinitas (affinity) adalah sejauh mana suatu
substansi cenderung ingin mengikat dengan
yang lain.
Prinsip kromatografi afinitas adalah
pengikatan spesifik ligan dengan target
molekul.
Jadi, dalam kromatografi afinitas minimum
harus ada dua senyawa yang berikatan
spesifik.
 Biological interactions between ligand and
target molecule can be a result of electrostatic
or hydrophobic interactions, van der Waals
forces and/or hydrogen bonding.
To elute the target molecule from the affnity
medium the interaction can be reversed,
either specifcally using a competitive ligand,
or non-specifcally, by changing the pH, ionic
strength or polarity.
 Some typical biological interactions, frequently used in
affnity chromatography, are listed below:
1. Enzyme substrate analogue, inhibitor, cofactor.
2. Antibody antigen, virus, cell.
3. Lectin polysaccharide, glycoprotein, cell surface
receptor, cell.
4. Nucleic acid complementary base sequence,
histones, nucleic acid polymerase, nucleic acid
binding protein.
5. Hormone, vitamin receptor, carrier protein.
6. Glutathione glutathione-S-transferase or GST
fusion proteins.
7. Metal ions Poly (His) fusion proteins, native
proteins with histidine, cysteine and/or tryptophan
residues on their surfaces.
COMPONENT OF STATIONER PHASE
 PREPARATION OF MEDIA AND BUFFERS

Use high quality water and chemicals.

Solutions should be filtered through 0.45
m or 0.22 m filters.
If an affinity medium is to be used routinely,
care must be taken to ensure that any
contaminants from the crude sample can be
removed by procedures that do not damage
the ligand.
 SAMPLE PREPARATION AND APPLICATION

Samples should be clear and free from particulate

matter.
If possible, test the affinity of the ligand:
target molecule interaction.
Too low affinity will result in poor yields since the
target protein may wash through or leak from the
column during sample application.
Too high affinity will result in low yields since the
target molecule may not dissociate from the
ligand during elution.
 For interactions with strong affnity between
the ligand and the target molecule that
quickly reach equilibrium, samples can be
applied at a high fow rate.
When working with very weak affinity
interactions that are slow to reach
equilibrium, it may be useful to stop the flow
after applying the sample to allow more time
for the interaction to take place before
continuing to wash the column.
 Do not begin elution of target substances until
all unbound material has been washed
through the column by the binding buffer
(determined by UV absorbance at 280 nm).
This will improve the purity of the eluted
target substance.
ELUTION
 ANALYSIS OF RESULTS AND FURTHER STEPS

The analysis of results from the first

separation can indicate if the purification
needs to be improved to increase the yield,
achieve higher purity, speed up the
separation or increase the amount of sample
that can be processed in a single run.
 Affinity
chromatography
The particular variant of
chromatography in which
the unique biological
specificity of the analyte
and ligand interaction is
utilized for the separation.

A specific molecule bound to the stationary phase is designed to react

to some specific analyte and bind to it.
It utilizes the specific interaction between one kind of solute molecule
and a second molecule that is immobilized on a stationary phase.
For example, the immobilized molecule may be an antibody to some
specific protein.
When solute containing a mixture of proteins are passed by this
molecule, only the specific protein is reacted to this antibody, binding it
to the stationary phase.
Affinity
Chromatrography
In affinity chromatography,
proteins are separated according
to their ability to bind to a
specific ligand that is connected
to the beads of the resin.
After the proteins that do not
bind the ligand are washed
through the column, the bound
protein of interest is eluted by a
solution containing free ligand.