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 µl well-suspended magnetic beads (10 mg/ml in lysis buffer) with 500 µl 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 µg 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, 4°C 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 µl bead suspension is about 4 µg 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 µl lysate (supernatant) add 400 µl BSA (10 mg/ml in lysis buffer).
  • Add 10 µg of the respective primary antibody and incubate at RT for 1 h
  • Suspend pretreated magnetic beads in 500 µl lysate solution and incubate at 4°C for 60 min using an overhead shaker
  • Pellet beads by magnetic force (or centrifugation)
  • Wash 5 times with lysis buffer
  • Elute with 40 µl 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!)