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Affinity Chromatography Explained In 3 Minutes
>> YOUR LINK HERE: ___ http://youtube.com/watch?v=PJVfNfmaWaA
0:00-0:56 | What is affinity chromatography? • 0:56-2:01 | Affinity chromatography workflow explained • 2:01-3:06 | Why use affinity chromatography? • Affinity chromatography is a separation method based on specific binding interaction between an immobilized ligand and its binding partner, such as antibody-antigen, enzyme-substrate and enzyme-inhibitor interactions. An nice analogy for this phenomenon is that of a key which specifically fits to a certain lock. The important point is that the ligand captures the wanted protein making it stick to it! Purification degree varies but can be quite high depending on specificity of the interaction used. • The workflow into 5 major steps: • 1. First cell lysate is added to the column • 2. The protein of interest will interact with the beads through matching affinity • 3. Add wash buffer and remove remaining unspecific protein and other substances • 4. Elute your protein of interest from the affinity beads through an elution buffer. Either via pH change or salt concentration which causes the protein of interest to unbind from the ligand • 5. Purified active protein of interest is collected! • So why is affinity chromatography so useful? Well, first of all it has a high selectivity, meaning that it is excellent at distinguishing between different proteins. It also has a high resolution meaning that there is a wide separation between the peaks in the results, allowing for easier analysis of the different compounds present in the sample. Finally it has a high capacity, meaning that the ligand is able to retain the protein of interest for a long time, allowing for easier separation.
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