Protocols:

 

Immunoprecipitation

 

of Phosphoserine-, Phosphothreonine- and Phosphotyrosine- Containing Proteins



We recommend immunoprecipitation with the use of magnetic beads.
Important Remarks:
  • Rinse all plastic vessels with BSA (10 mg/ml in lysis buffer).
  • For each immunoprecitation setup, pre-incubate 50 l well-suspended magnetic beads (10 mg/ml in lysis buffer) with 500 l BSA solution at RT for 30 min.
  • Pellet pre-incubated beads before use by magnetic force or centrifugation, discard supernatant.
  • Use lipid-free BSA, for example Sigma A7906.
  • Cell extracts contain a large number of phosphorylated proteins. Using antibodies directed against phospho amino acids, the precipitate will contain a variety of different phosphoproteins. In contrast to immunoprecipitation of single proteins, where less primary antibody is required, we recommend to use 10 g phospho amino acid specific primary antibody for immunoprecipitation of phosphoproteins from lysates of 106 cells.

1. Preparation of Cell Extracts:

Lysis Buffer:
20 mM Imidazole-HCl, pH 6.8
100 mM KCl
1 mM MgCl2
10 mM EGTA
0.2% (v/v) Triton X-100
10 mM NaF
1 mM Sodium Vanadate
1 mM Sodium Molybdate
10 mM Sodium Pyrophosphate
25 mM b-Glycerophosphate
Aliquote and store frozen!
  • suspend cells in cell lysis buffer (1ml buffer per 107 cells)
  • incubate at room temperature for 5 min (until cell lysis is terminated)
  • centrifuge at 14 000 g, 4C for 5 min. Use supernatant for immunoprecipitation. It is important to use freshly prepared supernatants for IP, since otherwise dephosphorylation may occur.

2. Pretreatment of Magnetic Beads:

We recommend antibody-conjugated magnetic beads from DYNAL:
for anti-PSER (clones 1C8, 4A9, 16B4, 4A3 and 4H4): anti-mouse IgM
for anti-PSER (clone 7F12): anti-mouse IgG
for anti-PTHR (clones 1E11 and 14B3): anti-mouse IgG
for anti-PTHR (clone 4D11): anti-mouse IgM
for anti-PTYR (clones 2C8, 3B12, 9H8 and 16F4): anti-mouse IgG

Binding capacity of 50 l bead suspension is about 4 g antibody. Beads can be purchased as 2 ml packaging units.


3. Immunoprecipitation Procedure:
  • Rinse all plastic vessels with BSA (10 mg/ml in lysis buffer)
  • For 100 l lysate (supernatant) add 400 l BSA (10 mg/ml in lysis buffer).
  • Add 10 g of the respective primary antibody and incubate at RT for 1 h
  • Suspend pretreated magnetic beads in 500 l lysate solution and incubate at 4C for 60 min using an overhead shaker
  • Pellet beads by magnetic force (or centrifugation)
  • Wash 5 times with lysis buffer
  • Elute with 40 l Laemmli sample buffer (SDS-PAGE sample buffer)
  • Perform SDS-PAGE with subsequent identification of proteins by fluorography or Western Blot (ECL system with peroxidase conjugate!)