基于色谱的生物大分子分离纯化方法

Size-exclusion chromatography separates molecules based on their size using a column packed with porous material. Large molecules cannot enter the pores and exit first, while small molecules enter the pores and exit last. Only molecules between the exclusion and permeation limits are separated from one another as they spend varying amounts of time diffusing in and out of the pores.

Molecules fall into three categories: those above the exclusion limit that cannot enter pores and exit together first; those below the permeation limit that freely enter pores and exit together last; and those between these limits that separate based on varying retention times. Molecular weights across one to two orders of magnitude typically fall within the separation range.

Affinity chromatography attaches target-specific ligands to the stationary phase. When a mixture flows through the column, target molecules selectively bind to the ligands while other components pass through. The target protein is then collected through elution methods that either compete with the ligand or change solution conditions like pH or ionic strength.

A polyhistidine tag is a short peptide sequence of repeating histidine residues engineered onto target proteins. In immobilized metal affinity chromatography, these tags bind to adhered metal ligands like nickel or cobalt via the imidazole side chain on histidine. The tagged protein is then released using free imidazole via reverse-role specific elution.

The IMAC procedure involves adding the stationary phase directly to the sample mixture to allow his-tagged protein binding. The slurry is poured into a column where unbound compounds drain to waste. After washing to remove unbound components, imidazole buffer is added to release the target protein by competing with the metal ligand.

Researchers combine these techniques to achieve higher purity levels. After affinity chromatography isolates the target protein using ligand binding, size-exclusion chromatography can further purify the sample by separating based on molecular size. This sequential approach is particularly useful when protein tags like polyhistidine may affect protein structure or function.

After fractions are collected from the size-exclusion column, gel electrophoresis and other analytical techniques are used to test the quality and identity of the target molecule in each fraction. This verification step ensures that the desired biomolecule has been successfully isolated and separated from other components in the mixture.