Immunoprecipitation

Question 1

Surface label

Answer 1

Biotinylating reagent

sulfo-LC-NHS-biotin

Question 2

Lysis buffer

Answer 2

20mM Tris-HCL, 150mM NaCl, 0.5% Nonidet P-40, 0.5% bovine serum albumin, 5 mM EDTA, 10ug/ml Leupeptin and 10ug/ml aprotinin, 0.5% Tween-20
Contains protease inhibitors but must still keep on ice to minimize proteolysis
Disrupts cell membrane but doesn’t interfere with Ag-Ab interactions

Question 3

Preclear

Answer 3

Goat serum-agarose beads

Coated w normal globulin or unrelated Ab from same sp. to remove materials that non-specifically bind to beads or IgG

Question 4

Agarose beads

Answer 4

Coupling of Ab to beads permits use of far less Ag and Ab than for direct precipitation from sol’n

Question 5

Aggregate precipitation

Answer 5

When enough antigen-antibody complexes are present, large aggregates may form. These aggregates may be pelleted by centrifugation at high speeds. Unfortunately, usually there is either not enough antigen and antibody to precipitate or too much cell debris for this to work

Question 6

Protein A and Protein G

Answer 6

Bacterial cell wall components that bind to Fc portion of antibodies.
Protein A is derived from the cell wall of Staphylococcus aureus
Protein G is derived from the cell wall of streptococci
Each different IP experiment can require unique antigen specific antibody-coated-beads but this can be expensive. Therefore, Protein A and Protein G are commonly coupled to agarose beads to allow for use with different antibodies rather than paying for each antibody to be conjugated to beads. Protein A or G coated beads can be used to isolate immune complexes.
NOTE***Protein A and Protein G are also used for antibody purification when isolating antibodies from culture supernatants in the preparation of large amounts of monoclonal antibodies.