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Immunoprecipitation (IP) and its derivative technique Co-immunoprecipitation (Co-IP) are core biochemical technologies based on antigen-antibody specific interaction, used for selectively enriching and purifying specific target proteins and their direct or indirect interacting molecular complexes from complex lysates. The core of this technology system is to immobilize antibodies on solid-phase substrates (such as agarose or magnetic beads), and through the principle of "affinity purification", efficiently capture target antigens from thousands of protein backgrounds for subsequent qualitative, quantitative, and functional analysis.

You can choose a standard operation procedure that matches your research materials for cell or tissue lysis. You can also refer to the sample lysis operation in the "Western Blot" operation on the HUABIO official website.

(1) High concentration of detergent will interfere with immunoprecipitation effect. So you can first use RIPA to lyse cells or tissues to prepare protein lysate, and then add 1xTBS or IP incubation buffer to dilute to the required volume at the beginning of the immunoprecipitation step.

(2) Use sufficient protein lysate: For single-tube IP experiment, the recommended initial protein amount of lysate is 1-3 mg.

(3) Ensure that protease inhibitors are added to the lysate, and the dosage of protease inhibitors can be 1.5-2 times that of normal WB experiment sample preparation.

Experimental Applications

Immunoprecipitation (IP) Operation Procedure

1. Add an appropriate amount of lysate to an EP tube (low adsorption), add 1-4μg antibody at the same time, make up to 1mL volume with 1xTBS or IP incubation buffer, and the optimal antibody amount should be determined by antibody titer. Take another equal amount of lysate, add equal amount of 1xTBS or IP incubation buffer and equal amount of isotype IgG as Control IgG control group.

2. Place all EP tubes on a rotating rack, 4°C, rotate for 2–4h or overnight.

3. Add 20-50μl resuspended Protein A agarose beads or magnetic beads, incubate at 4°C with rotation for 1—2h.

4. Centrifuge at 500g for 30s at 4°C, discard supernatant. Or place on a magnetic rack and let stand for 1 minute.

5. Add 1mL of 1×TBS containing protease inhibitors, wash the precipitate complex, centrifuge at 500g for 30s at 4°C (or place on a magnetic rack and let stand for 1 minute), aspirate and discard supernatant; repeat washing 4—5 times. After the last washing/centrifugation, keep about 80 μl solution in the EP tube (discard the rest).

6. Add 20μl 5x SDS-PAGE protein loading buffer, resuspend IP complex beads, boil at 95–100°C for 5 minutes, then centrifuge at 8,000-10,000g for 3 minutes, carefully aspirate the supernatant and transfer to a new EP tube.

7. Add the collected IP sample to the corresponding lane of SDS-PAGE, and the remaining IP sample can be stored at -80°C for later use.

8. Separate IP samples by SDS-PAGE, transfer proteins to PVDF membrane, and use appropriate antibodies for subsequent detection.

Note: When performing WB detection on IP samples, to eliminate or reduce the interference of antibody heavy and light chains in the sample, IP secondary antibodies can be used for detection, such as HUABIO Catalog# NBI01H (rabbit) and Catalog# NBI02H (mouse).