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Standard Protocol for Immunoprecipitation

Solutions needed

  • Solubilization buffer A: 10 mM Tris/HCl (pH 7.4), 50 mM NaCl, 2% Triton-X100, Protease inhibitors
  • Solubilization buffer B (optional):
  • Solubilization buffer C (optional):
  • Buffer A: 10 mM Tris/HCl (pH 7.4), Protease inhibitors
  • Buffer B: 10 mM Tris/HCl (pH 7.4), 150 mM NaCl, 0,2% Triton X100, Protease inhibitors
  • Blocking buffer: 10 mM Tris/HCl (pH 7.4), 2% BSA, Protease inhibitors

Procedure

All steps should be carried out at 4 °C. Protein solubilization (Solubilization step 1) and binding of the antibodies to the beads (Immunoprecipitation step 1) can be carried out in parallel.
Protease inhibitors should be included.

Solubilization with Triton-X100

  1. Solubilize proteins by adding a 1:1 volume of Solubilization buffer A to the cell or tissue sample (final concentration of 5 mM Tris/HCl, 25 mM NaCl, 1% Triton-X100).
    Starting material should have a concentration of 1 - 4 mg/ml.
  2. Centrifuge unsolubilized material at 9600 x g for 10 min. The supernatant will be used for IP. If desired, the pellet can be kept for further analysis.

Denaturing solubilization with SDS

  1. Adjust protein samples to 3 mg/ml total protein and a final SDS concentration of 1.2% with solubilization buffer B and rotate 15 min at RT.
  2. Add 5 volumes of ice-cold solubilization buffer C to each sample and rotate 15 min at 4°C.
  3. Pellet the insoluble fraction at 100,000 x g for 30 min (acceptable alternative: 13,000 rpm for 30 min at 4°C in a tabletop centrifuge) and transfer the supernatant to a new tube. Dilute the supernatant in antigen buffer B to 0.2% Triton concentration.
    Note: If complete tissue samples are used, DNase should be added as 0.1 µg/µl together with protease inhibitors, and SDS should be added as last component after mixing everything else.

Immunoprecipitation

  1. Incubate 5 - 10 µg of antibody or 5 µl antiserum with 10 µl Protein G or A slurry in 200 µl buffer A for 1 h to bind.
  2. Centrifuge beads for 5 min at 2400 x g and discard the supernatant.
  3. Block beads with 200 µl of blocking buffer for 30 min.
  4. Centrifuge beads for 5 min at 2400 x g and discard the supernatant.
  5. Wash beads with buffer A, centrifuge beads for 5 min at 2400 x g, and carefully remove buffer A.
  6. Add 100 - 200 µl of the sample and incubate for 1 - 2 h at 4°C rotating head over tail.
  7. Centrifuge beads for 5 min at 2400 x g and collect supernatant for subsequent analysis.
  8. Wash suspension twice with buffer B. Centrifuge beads for 5 min at 2400 x g and remove buffer B.
  9. For SDS-PAGE analysis, incubate the pellet with SDS loading buffer and apply to SDS-PAGE. Apply starting material and supernatant from step 9 for comparison.
 

Remarks

  • Some proteins are not efficiently solubilized by triton. For these proteins, the denaturing solubilization protocol is recommended. For further details: Geumann et al. should be employed: Geumann C, Grønborg M, Hellwig M, Martens H & Jahn R (2010). A sandwich enzyme-linked immunosorbent assay for the quantification of insoluble membrane and scaffold proteins. Analytical Biochemistry 402: 161-9.
  • If membrane proteins are immunoprecipitated, make sure that detergent is included in all steps that contain your target protein. 
  • Not all IgG subtypes from all species bind equally well to protein A or protein G. It is therefore important to choose the right resin (for details see table 1).
     
    Species Subclass Protein A
    binding
    Protein G binding
    Human IgA variable -
      IgD - -
      IgE - -
      IgG1 ++++ ++++
      IgG2 ++++ ++++
      IgG3 - ++++
      IgG4 ++++ ++++
      IgM variable -
    Avian egg yolk IgY - -
    Cow IgG ++ ++++
    Dog IgG ++ +
    Goat IgG - ++
    Guinea pig IgG ++++ ++
    Hamster IgG + ++
    Horse IgG ++ ++++
    Llama IgG - +
    Monkey (rhesus) IgG ++++ ++++
    Mouse IgG1 + ++++
      IgG2a ++++ ++++
      IgG2b +++ +++
      IgG3 ++ +++
      IgM - -
    Pig IgG +++ +++
    Rabbit IgG1 ++++ +++
    Rat IgG1 - +
      IgG2a - ++++
      IgG2b - ++
      IgG3 + ++
    Sheep IgG +/- ++
                                                                             Table 1