Immunoprecipitation (IP)

Immunoprecipitation

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Crosslink immunoprecipitation (IP)

Materials and method

Materials

• Protein A/G plus Agarose – protein A/G are used with rabbit/mouse antibodies respectively
• 20X Coupling buffer
• Disuccinimidyl suberate (DSS)
• IP Lysis/Wash buffer
• 100X Conditioning Buffer
• 20X Tris-Buffered Saline
• Elution Buffer
• Non-reducing 5X Lane Marker Sample Buffer
• Spin Columns
• Microcentrifuge Collection Tubes
• Microcentrifuge Sample Tubes
• Control Agarose Resin – used to pre-clear lysates to reduce non-specific protein binding
• Ultrapure water

Note:

• Flow through rate should not exceed 600/300ml when using a 2/1.5ml collection tube respectively; as exceeding these volume may result in back pressure in the spin column/incomplete wash/incomplete elution.
• All resin centrifugation steps should be performed at low speeds i.e. 1000-3000 x g as high-speed centrifugation i.e. > 5000 x g may cause resin to clump and make resuspension difficult.

Method

(i) Binding of antibody to protein A/G plus Agarose

1. Prepare 2ml of 1X coupling buffer for each IP reaction by dilution 20X coupling buffer with ultrapure water
2. Using a cut pipette tip, add 20ml of resin slurry into a spin column and place the column into a microcentrifuge tube and centrifuge at 1000 x g for 1 minute. Then discard the flow through. Note: Gently swirl the bottle of pierce protein A/G plus agarose to obtain an even suspension
3. Wash the resin twice with 200ml of 1X coupling buffer, centrifuge and discard the flow-through.
4. Gently tap the bottom of the column on a paper towel to remove excess liquid and insert the bottom plug.
5. Prepare 10mg of antibody for coupling by adjusting the volume to 100ml with sufficient volume of ultrapure water and 20X coupling bluffer. Note: Add the ultrapure water, 20X coupling buffer and affinity-purified antibody directly to the resin column.
6. Attach the screw cap to the column and incubate on the rotator at room temperature for 30-60minutes; ensuring that the slurry remains suspended during incubation.
7. Remove and retain the bottom plug and remove the cap. Then place the column in a collection tube and centrifuge. Note: Save the flow-through to verify antibody coupling.
8. Wash the resin with 100ml of 1X coupling buffer, centrifuge and discard the flow-through.
9. Wash the resin twice with 300ml of 1X coupling buffer, centrifuge and discard the flow-through.

(ii) Crosslinking of bound antibody
Note: DSS crosslinker is moisture sensitive and incompatible with amine-containing buffers e.g. Tris, glycine. DSS should also be dissolved in DMSO/DMF immediately before use (dilute DSS solution in 1:10 DMSO/DMF to make 2.5mM)

1. Tap the bottom of the column on a paper towel to remove excess liquid and insert the bottom plug.
2. Add 2.5ml of 20X coupling buffer, 9ml of 2.5mM DSS and 38.5ml of ultrapure water to the column to make up 50ml. Then attach the screw cap.
3. Incubate the crosslinking reaction or 30-60 minutes at room temperature on a rotator.
4. Remove and retain the bottom plug and open the bottom plug and screw cap and place the coloumn into a collection tube and centrifuge.
5. Add 50ml of elution buffer to the column and centrifuge. Note: Save the flow-through to verify antibody crosslinking.
6. Wash four times with 200ml of elution buffer to remove non-crosslinked antibody and quench the crosslinking reaction.
7. Wash twice with 200ml of cold IP lysis/wash buffer and centrifuge after each wash. Note: Antibody-crosslinked resin can be stored up to 5 days in IP lysis/wash buffer. For longer storage, store resin in 1X coupling buffer (PBS).

(iii) Antigen immunoprecipitation

Note: If antibody-crosslinked resin was stored in PBS, wash twice with IP lysis/wash buffer and discard the flow-through after each wash.

1. Tap the bottom of the column on a paper towel to remove excess liquid and replace the bottom plug.
2. Add sample to the antibody-crosslinked resin in the column. Then attach the screw cap and incubate the column overnight at 4^oC on the rotator.
3. Remove the bottom plug, loosen the screw cap and place in the column in a collection tube and centrifuge the column. Save the flow through. Note: Do not discard the flow-through until confirming that immunoprecipitatio is successful.
4. Remove the screw cap, and place the column into a new tube. Add 200ml of 1X TBS buffer and centrifuge.
5. Wash sample twice with 200ml IP lysis/wash buffer and centrifuge after each wash.
6. Wash sample once with 100ml of 1X conditioning buffer.

(iv) Antigen elution

1. Add 20ml of elution buffer into the column and incubate for 5minutes at room temperature. Centrifuge.