Immunoprecipitation and chromatography by k5V4cU


									  Immunoprecipitation and
Techniques used to purify (separate)
a specific protein from a mixture of
          • Purpose:
          • To isolate a specific protein
            (antigen) from a mixture of

          • First, add antibody which
            will bind to antigen
          • Then add beads complexed
            with a second antibody that
            will bind to the first
          • Lastly, a magnet will pull
            the complex out of solution
            and you will have isolated
            your protein
• 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
• 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
Separate proteins based on mass, charge or
             binding affinity
• Gel filtration chromatography – size

• Ion-exchange chromatography – charge

• Affinity chromatography – binding to a
  specific molecule
    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
        Ion exchange and affinity
• 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
    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
   – Decrease affinity of protein for the stationary phase and
     increase affinity for the mobile phase
        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
To determine progress of chromatography
• Take different fractions
• Read absorbance at 280nm – maximal protein

• Or: take fractions, and perform
  – Initially, many proteins, later on less proteins

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