Immunoprecipitation and chromatography

1. Immunoprecipitation and chromatography Techniques used to purify (separate) a specific protein from a mixture of proteins

2. Immunoprecipitation Purpose: To isolate a specific protein (antigen) from a mixture of proteins First, add antibody which will bind to antigen Then add beads complexed with a second antibody that will bind to the first antibody Lastly, a magnet will pull the complex out of solution and you will have isolated your protein

3. Chromatography Add a mixture of proteins in a mobile phase to some sort of stationary phase Some proteins have high affinity for the mobile phase and will move quickly through the column Some proteins have a high affinity for the stationary phase and will move slowly through the stationary phase and stick to the column At the end of the procedure, conditions are changed inorder to elute the protein from the column

4. Separate proteins based on mass, charge or binding affinity Gel filtration chromatography – size Ion-exchange chromatography – charge Affinity chromatography – binding to a specific molecule

5. Gel filtration chromatography Separate based on size Use column of beads made of polyacrylamide, dextran or agarose Smaller proteins can penetrate the beads more easily than larger proteins Smaller proteins trapped in the column; larger molecules come off the column first Need more volume to elute the smaller proteins Compare to standards of known molecular weight to determine the mass of an unknown protein

6. Ion exchange and affinity chromatography The column has some affinity for binding certain proteins Charge or a certain ligand is bound to the column To elute the protein: Increase the salt concentration = decreases the affinity for the column change the pH – will change the charge of the protein

7. Ion exchange chromatography Separates proteins that differ in charge Cation exchange column is negatively charged Binds positively charge proteins Anion exchange column is positively charged Binds negatively charged proteins Elute by increasing the salt concentration This changes the affinity of your protein for the different phases: Decrease affinity of protein for the stationary phase and increase affinity for the mobile phase

8. Affinity chromatography Separation of proteins based on their ability to bind to another molecule Use an antibody to isolate corresponding antigenic proteins from a mixture of proteins Covalently link a reagent to a column, only proteins that bind the reagent will be retained by the column, all others will pass through The proteins that are bound to the column are eluted by adding an excess of ligand or by changing the salt concentration or pH

9. To determine progress of chromatography separation Take different fractions Read absorbance at 280nm – maximal protein absorbance Or: take fractions, and perform electrophoresis Initially, many proteins, later on less proteins