The Principle Of Affinity Chromatography

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The objective of this lab was to learn the principle of affinity chromatography by isolating a carbohydrate-binding protein from an extract of jack bean meal.

The protein that was used for this lab came from jack bean meal, and it is identified as Concanavalin A, or Con-A. This is a lectin protein that has the ability to bind with glucose, but it is not a glycoprotein. Furthermore, the monomeric molecular weight of this protein is 25,5001. Concanavalin A also exists as a dimer and a tetramer, and this depends upon the pH of this substance. Finally, Con-A requires a transition metal ion such as manganese as well as calcium ions for binding purposes.

1. Begin by inserting a cheesecloth into a syringe and pressing the cheesecloth to the very bottom of the column.
2. Pour 10 ml of the affinity gel into the column. Allow a few minutes for the solution to harden and transform into a gel-like consistency.
3. Fill the rest of the column with NaCl, and do so by pouring the solution along the interior side of the column. This allows the gel to become moist.
4. Next, gather 0.5 ml of the protein extract and save this portion in a separate container. This will be used for the preparation of the membrane.
5. Load the remaining protein mix into the column, and this will begin to bind within the column. Once the solution has settled, fill the column with NaCl a total of four times; as the wash is flowing through, collect and label the protein solution in fractions, and save for the
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