Immunoprecipitation (IP) –1. Cell Lysis, Pre-clearing

What Is Immunoprecipitation?

Immunoprecipitation is used to precipitate a protein antigen out of solution using an antibody (antibody-protein)

Co-immunoprecipitation is the IP of intact protein complexes (protein-protein-antibody).

Depending on the purpose, it can be called IP (antibody-protein), co-IP (protein-protein-antibody), RIP (RNA-protein-antibody), ChIP (DNA-protein-antibody).

Simple Procedure

Lysis sample → Add antibody in the lysate → add protein agarose beads → precipitate → elution.

To reduce background, I recommend you do pre-clearing the lysate to remove proteins that binds to protein beads non-specifically. If you are planning to detect by western blotting, pre-clearing step is not required unless a contaminating protein is interfering with visualization of the protein of interest.

Cell Lysis

Lysis Buffer for IP

The ideal lysis buffer will minimize denaturation of antibody binding sites while releasing the proteins from the sample. RIPA buffer without SDS or NP-40 lysis buffer are widely used as lysis buffer for IP. I’ve used RIPA buffer with 0.1% SDS but still was able to get good results.

* Protease inhibitor: cOmplete™ Protease Inhibitor Cocktail, Roche

Role of Each Components

Tris-HCl: pH adjustment

NaCl: keeps protein soluble and increases the ionic strength of the buffer, which disrupts molecular interactions.

NP-40: non-ionic detergent for lipid membrane dissociation

sodium deoxycholate: ionic detergent (can dissociate membrane easily but can destroy protein too)

SDS: anionic detergent (can dissociate membrane easily but can destroy protein too)

Protease and phosphatase inhibitor: inhibits the proteolysis, dephosphorylation, denaturation (for the same reason, lysis the samples on ice).

Cell Lysis

1. Wash one 80-90% confluency of 10 cm dish (~8X10^6 cells) with cold PBS, twice. Note. To extract protein, use cold PBS instead of warm PBS. Cooling the cells down reduces cell shock but also reduces protease activities.

2. Add 1 ml of cold PBS and scrape the cells with a scraper.

3. Transfer to an ep-tube

4. Add 0.5 ml of cold PBS to get cells as much as possible and transfer to the ep-tube. Keep on ice.

5. Centrifuge at 12,000g for 5 min at 4°C.

6. Remove sup as much as possible.

7. Add 100 ul (depends on the pellet size) of 1X RIPA buffer to the sample tube.

8. Mix by pipetting and vortex for 10 sec.

9. Incubate on ice for 1 hr.

10. (Option) Vortex 10 sec again and incubate 1 more hour on ice. Note. To get higher yield, I’ve used to store the cells in lysis buffer at -80°C overnight when I had enough time.

11. Sonicate using sonication for 10 cycles, 30 sec on/ 60 sec off, medium power.

12. Fill more ice in the chamber. Note. To prevent excessive heating causing protein degradation, make sure that enough ice is in the chamber.

13. Sonicate for 10 cycles, 30 sec on/ 60 sec off, medium power.

14. Centrifuge at 12,000g for 10 min at 4°C.

15. Take the supernatant. Note. Lysate can be stored at -20°C for up to 3 months. For long term storage, keep at -80°C.

Pre-Clearing

1. Add 0.4 ug of normal IgG into 500-700 ug of lysate. Note1. The host of normal IgG has to be same as the host of antibody for IP. For example, if you are using a mouse antibody for IP, use mouse normal IgG for preclearing. Note2. The purpose of adding IgG is to remove proteins non-specifically bind to antibody.

2. Add 20 ul of protein agarose beads (Santa cruz, cat.SC-2003). Note1. See Selection of Agarose Beads. Note2. Use wide bore tip or cut the end of pipette tip to take agarose beads.

3. Add lysis buffer to adjust volume. Note1. A good rule of thumb is to make the final volume of 500-600 ul. Note2. The reason adjusting volume is to make sure that sample can be agitated properly.

4. Rotate for 1 h at 4°C. Note. Must be processed at 4°C. Minimum 30 min of pre-clearing is required.

5. Centrifuge at 1,200g for 1 min at 4°C.

6. Take sup. This is the pre-cleared lysate.

Selection of Agarose Beads. For IP, using agarose beads or magnetic beads are the most common systems.

Agarose beads are cheaper than magnetic beads and have higher antibody binding affinity. But magnet beads system is easier to work as it requires not only fewer working steps but also shorter incubation time. If you want to do IP using agarose beads, you have to choice one among protein G agarose beads, protein A agarose beads, and protein A/G agarose beads.

Both protein A and protein G have high affinity for the Fc region of IgG. They are structurally very similar, but they have slightly different affinities for IgG subclasses. Thus, Protein A has higher affinity for rabbit, dog, guinea pig, rabbit, pig, and cat IgG whereas protein G has higher affinity for mouse, human, cow, goat, hamster, horse, rat, and sheep. Protein A/G is the protein overcame the limit of Protein A or Protein G. Protein A/G binds nearly any mammalian IgG.