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

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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