Classic Immunoprecipitation (IP) Protocol

Protocol

A. Lysis of a Mammalian Cell Suspension

1. Centrifuge the cell suspension at 2,500 × g for 10 minutes to pellet the cells. Discard the supernatant. 
2. Optional wash: If the culture medium contained phenol red or other reagents that could interfere with subsequent protein analysis, wash the cells once by resuspending the cell pellet in a desired wash buffer (e.g., PBS). Centrifuge at 2,500 × g for 10 minutes to pellet the cells.
3. Add M-PER Reagent to the cell pellet (500 µl of M-PER Reagent is sufficient for lysing 50 mg of wet cell pellet). For optimal results use a 10:1 v/w ratio. Note: If using a large amount of cells, first add 10% of the final volume of M-PER Reagent to the cell pellet. Pipette the mixture to suspend pellet. Add the remaining volume of the M-PER Reagent to the cell suspension. 
4. Shake the reaction gently for 10 minutes. Remove cell debris by centrifugation at 27,000 × g for 15 minutes and transfer supernatant to a new tube for further analysis.

B. Preparation of Immune Complex

•The amount of antigen needed and the incubation time are dependent upon the antibody-antigen system used and will have to be optimized for each specific system.
• For optimal results, use an affinity-purified antibody. Although serum can be used, the antibody that is specific for the antigen of interest may comprise only 1-2% of the total IgG in the serum sample and will result in low antigen yields.
• On average, rabbit serum contains ~10-12 mg/ml IgG and mouse contains ~6-8 mg/ml IgG. Some species may contain up to 30 mg/ml IgG. Ascites fluid contains an average IgG concentration of ~1-10 mg/ml. 
• Tissue culture supernatant (serum-free medium) contains an average of 0.05 mg/ml IgG. This amount may be too dilute to effectively capture enough antigen for analysis. For best results concentrate tissue culture supernatant to at least 500 µg/ml before forming the immune complex.
1. Prepare 0.1-1.2 mg of purified antibody at a concentration of 0.2-10 mg/ml. Use a volume of 0.1-5 ml. 
2. Add purified antibody to sample or lysate. Incubate the sample/lysate with the antibody for at least 30 minutes. For overnight incubations, incubate sample at 4ºC.

C. Equilibration of Protein G Agarose Beads.

1. Equilibrate the Protein G Agarose and reagents to room temperature.
2. Add 500 ml of ultrapure water to the Modified Dulbecco's PBS buffer pack contents. To store excess buffer, add a preservative such as 0.02% sodium azide and store at 4°C.
3. Gently swirl the bottle of Protein G beads to obtain an even suspension. Add 0.4 ml of the 50% bead slurry into one of the Pierce Spin Cup Columns and place inside a Pierce Microcentrifuge Tube. 
4. Centrifuge the tube and discard flow-through. Place the spin cup back into the tube. 
5. Wash the agarose beads by adding 0.4 ml of PBS to the spin cup containing the resin. Cap the tube and gently mix end over end or place on a rocker for ~1-2 minutes. Centrifuge the tube and discard flow-through. Replace the spin cup into the tube. Repeat this wash once and place the spin cup into a new microcentrifuge tube.

D. Capture of the Immune Complex

1. Add immune complex prepared in Section A to the spin cup containing the equilibrated Protein G beads and cap the tube. (Recommended loading is 0.05-0.4 ml.) Incubate for at least 10 minutes. Centrifuge the tube and discard flow through. Repeat this step until the entire sample has been applied. 
Note: The flow-through may be reserved in another container until the IP has been completed. This precaution may be useful if the antibody/antigen complex did not bind well to the Protein G.
2. Add 0.5 ml of PBS into the spin cup. Cap the tube and invert it 5-10 times. Centrifuge the tube and discard flow-through or save it in a separate tube for analysis. Repeat wash step at least two additional times using the same collection tube. After the final wash, place the spin cup into a new tube and wash one additional time.
Note: To avoid contamination from residual proteins, before eluting the complex verify that the resin has been thoroughly washed by performing a protein assay on the flow-through from the final wash. There should be minimal protein in the final wash fraction. If the material has not been adequately washed, repeat Step 2 before proceeding. Additional washes may be necessary for samples containing high protein concentrations.

E. Elution of the Immune Complex 

Note: Before using the purified material in applications requiring functional protein, neutralize the sample after eluting the complex. The Elution Buffer has a pH of 2.5-3.0 and can be neutralized by adding 10 µl of 1 M Tris, pH 9.5 per 200 µl of eluted sample. Alternatively, if the protein is sensitive to low pH, try using a neutral pH system such as the Gentle Elution Buffer. If performing SDS-PAGE, it is not necessary to neutralize the eluted sample. However, the dye in the sample buffer may change color caused by the low pH, but this color change will disappear after the sample enters the electrophoresis gel.
1. Add 190 µl of Elution Buffer to the spin cup containing the immune complex. Cap tube and invert it 10 times. Centrifuge the tube and place spin cup into a new tube.
Note: Smaller elution volumes may be used if the contents of the spin cup are first transferred to a microcentrifuge tube. Elution volumes must be sufficient to resuspend the resin beads. 
2. Repeat Step 1 until desired sample is eluted. Samples will usually elute within the first three fractions. Do not pool fractions. Assess the first three fractions by SDS-PAGE. 
3. To preserve activity of the Protein G, proceed to Section E immediately following the last elution step.

F. Agarose Bead Regeneration and Storage Conditions 

1. Add 0.5 ml of PBS to the spin cup. Cap tube and invert it 10 times. Centrifuge the tube and discard flow-through. Repeat this step once. 
2. Add 0.5 ml of PBS to the spin cup. Place the spin cup into a microcentrifuge tube, cap tube and wrap the cap with laboratory film to prevent drying. For long-term storage, add sodium azide (0.02% final). 
3. For convenience, place the capped tube into the foam insert of the kit box and store at 4°C. Note: A sufficient number of microcentrifuge tubes is supplied for ~50 IPs if the storage tube is also used during the wash steps of the IP procedure. If more tubes are required, any 1.5 ml microcentrifuge tube may be substituted. 

G. Preparation of Samples for SDS-PAGE 

1. Add 20 µl of the sample to a microcentrifuge tube.
2. Equilibrate the Lane Marker Sample Buffer (dark pink solution) to room temperature. Gently mix the Sample Buffer by inverting 5-10 times.
Pipette 5 µl of the 5X Sample Buffer into the microcentrifuge tube. 
Note: The sample buffer is viscous and may require that the pipette tip be "snipped" to allow the solution to be drawn into the tip. Pipette up and down to mix. This sample buffer does not contain reducing agents. To prepare the sample for a reducing gel, add 2-3 µl of 1 M DTT (MW =154.25) to the 25 µl sample containing sample buffer and mix well. Sample buffer from other vendors may also be used.
3. Insert tube into a microcentrifuge tube holder and place it in boiling water. Boil sample for ~5 minutes. 
4. Allow the sample to cool to room temperature and apply to the gel for electrophoresis.