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	+/-	++