Kromatografi Afinitas

Indah Solihah
 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.
 Other Function
• Affnity chromatography is also used to remove
specifc contaminants, for example
Benzamidine Sepharose™ 6 Fast Flow can
remove serine proteases, such as thrombin
and Factor Xa.
Component of stationer phase
 Media selection
• A ligand already coupled to a matrix is the
simplest solution.
• If a ligand is available, but needs to be coupled
to a pre-activated matrix.
• If no suitable ligand is available, it may be
more convenient to use alternative purifcation
techniques such as ion exchange or
hydrophobic interaction chromatography
 Preparation of media and buffers
• Use high quality water and chemicals.
Solutions should be fltered through 0.45 µm
or 0.22 µm flters.
• If an affnity 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 affnity of the ligand: target
molecule interaction.
• Too low affnity will result in poor yields since the
target protein may wash through or leak from the
column during sample application.
• Too high affnity 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 affnity
interactions that are slow to reach
equilibrium, it may be useful to stop the fow
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 frst separation
can indicate if the purifcation 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.