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.
