IP PROTOCOL

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Immunoprecipitation (IP) is an affinity purification technique that utilizes the characteristic binding of antibodies to antigens, and is widely used to enrich and isolate natural target proteins from tissue or cell lysates.

Note: Keep specimens on ice or at 4°C whenever possible.

I. Sample Preparation

Cell lysis can be performed by selecting a standard procedure that is appropriate for the material being studied.

Tip 1: High concentration of desalting agents may interfere with the immunoprecipitation effect. Therefore, protein lysates can be prepared by lysing cells with a small volume of RIPA and then diluting to the desired volume with 1 X PBS or incubation solution before starting IP capture.

Tip 2: Use a sufficient amount of protein lysate: For single-tube IP experiments, the recommended starting volume of lysate is 0.2-0.4 ml, containing 1-3 mg of total protein. The total protein concentration can be determined by the Bradford or BCA method.

Tip 3: Ensure that protease inhibitors are added to the lysate. The amount of protease inhibitor can be 1.5-2 times that of the normal WB sample.

Lysate Pretreatment (optional)

l Cut the end of the sterilized tip at a 45° angle and rapidly aspirate the resuspended Protein A Sepharose bead suspension into the EP tube containing the lysate. Typically, 1-3 mg of total protein lysate is added to 30 μL of resuspended Protein A Sepharose beads suspension.

Tip 4: Cut the end of the sterilized tip at a 45° angle to prevent beads from clogging the tip and causing uneven aspiration.

l Incubate at 4°C for 60 minutes with rotation (vertical shaker recommended, low speed rotation).

l Centrifuge at 4°C, 500g for 1 minute and transfer the supernatant into a new EP tube.

Tip 5: For IgG-rich tissues, a pretreatment step with Protein A beads or Protein G beads is recommended.

II. Immunoprecipitation

1. Aspirate 200-400 μL lysate (or pretreatment) containing 1-3 mg total protein into an EP tube and add 1-4 μg specific antibody and 150-300 μL incubation solution, the optimal amount of antibody should be determined by antibody potency. An equal amount of protein lysate was also taken and incubated with an equal amount of incubation solution and an equal amount of isotype IgG as a control IgG. rotate at 4°C for 2-4 hours or overnight.

2. Then add 50 µl of resuspended Protein A-Agarose beads, rotate and incubate at 4°C for 1-4 hours.

3. Centrifuge at 4°C, 500g for 30 seconds and discard the supernatant.

4. Wash the precipitated complex with 1 mL each of 1× TBST, now with protease inhibitor, and centrifuge at 4°C, 500g for 30 s. Aspirate the supernatant; repeat the washing 4-5 times. At the end of the last wash/centrifugation, approximately 80 ul of solution will remain in the EP tube (the remainder is aspirated).

5. Add 20 μL of 5x SDS Sample Buffer, resuspend the IP complex beads, boil at 95-100°C for 5 minutes, centrifuge at 8,000-10,000g for 3 minutes, and transfer the supernatant to a new EP tube.

III. WB Analysis

1. The collected IP samples are added to the appropriate lane of the SDS-PAGE, and the remaining IP samples can also be stored at -80℃.

2. The IP samples were separated by SDS-PAGE and the proteins were transferred to the PVDF membrane. Subsequent detection was performed using appropriate antibodies.

Tip 6: To eliminate or minimize interference from heavy and light chain antibodies in IP samples, HRP-anti-rabbit light chain specific secondary antibody or HRP-Protein A can be used instead of the traditional WB secondary antibody for WB detection. (HRP-Protein A has better affinity for natural intact antibodies than denatured antibodies).

IV. How to select the detection of secondary antibody

IP Capture Antibody Type	Primary antibody type for WB assay	Type of secondary antibody for WB assay	Commentary
Mouse monoclonal antibody/polyclonal antibody	Mouse monoclonal antibody	HRP-labeled Protein A or HRP-labeled anti-mouse IgG secondary antibody	HRP-conjugated anti-mouse IgG secondary antibody may cause strong interference signals from heavy chain and light chain in WB assay; HRP-conjugated protein A can effectively reduce the intensity of heavy chain signals and attenuate light chain signals; If the primary antibody of the WB is mouse IgG1/IgG3 isoforms, the affinity of HRP-conjugated protein A is relatively low, and the dilution of HRP-conjugated protein A can be decreased accordingly (or we can try to select HRP-conjugated anti-mouse IgG secondary antibody). If the WB primary antibody is mouse IgG1/IgG3 subtype, the affinity of HRP-conjugated Protein A is low, and its dilution can be decreased accordingly (or we can try HRP-conjugated anti-mouse IgG secondary antibody first).
	Mousepolyclonalantibody
	Rabbit antibody	HRP-labeled anti-rabbit IgG secondary antibody	Since the IP capture antibody and the WB primary antibody are antibodies of different origin, the use of HRP-conjugated anti-rabbit IgG secondary antibody in the WB assay can effectively avoid the influence of heavy chain and light chain signals.
Rabbit antibody	Mouse monoclonal antibody	HRP-labeled anti-mouse IgG secondary antibody	Because the IP capture antibody and the WB primary antibody belong to different types of antibodies, the WB test The use of HRP-conjugated anti-mouse IgG secondary antibody can effectively avoid the influence of heavy chain and light chain signals.
	Mousepolyclonalantibody
	Rabbit antibody	1, HRP-labeled protein A 2, HRP-conjugated anti-rabbit IgG light chain specific antibody	1、HRP-labeled anti-rabbit IgG secondary antibody will produce strong heavy chain and light chain signals and background signals during WB detection, which will have a certain influence on the analysis of the results; 2、HRP-labeled Protein A can effectively reduce the heavy chain signal as well as attenuate the influence of light chain signal, clean background, suitable for detecting all target proteins except 45-55 kDa, and when the target protein size is between 45-55 kDa, we recommend the use of HRP-labeled anti-rabbit IgG light chain specific secondary antibody.