Immunoprecipitation

Immunoprecipitation (IP) is a technique that uses specific antibodies to bind antigens and enriches target proteins from complex samples via precipitation. It is applicable for detecting interactions between target proteins and other proteins or DNA fragments. IP techniques include: Direct IP (enrichment of a single protein), Co-Immunoprecipitation (Co-IP, for studying protein-protein interactions) and Chromatin Immunoprecipitation (ChIP, for studying DNA-protein interactions)^[1].

This protocol focuses on “narrow-sense IP”—isolating and concentrating specific proteins from samples containing multiple proteins^[1].

Technical Principle: The core of IP relies on the highly specific antigen-antibody binding. When antibodies are conjugated to a solid-phase matrix (e.g., agarose beads or magnetic beads), they form an “antibody-antigen-binding protein complex” with target proteins in the solution. Specific enrichment is achieved via centrifugation (for agarose beads) or magnetic separation (for magnetic beads). Co-IP further extends this principle: by capturing one protein, it indirectly isolates other proteins that have physical interactions with it. These interactions can be direct (e.g., enzyme-substrate) or indirect (e.g., proteins in the same complex)^[2].

Development and Applications: IP has multiple technical variants, including competitive Co-IP, functional Co-IP, and Affinity Purification-Mass Spectrometry (AP-MS). As a key tool in proteomics, IP has expanded from simple binary interaction verification to high-throughput analysis of complex protein machines — driven by advances in antibody engineering and mass spectrometry. Future integration with emerging technologies (e.g., super-resolution microscopy, microfluidic platforms) will further promote its use in dynamic interaction research and clinical diagnostics^[3].

Co-IP experiments are complex and require not only high-quality antibodies but also experienced operating skills. MCE provides you with high-quality immunoprecipitation-specific reagents and professional technical support services. For more service information, please visit the official website: https://www.medchemexpress.com/

MCE has not independently verified the accuracy of these methods. They are for reference only.

The following steps describe IP using cell samples as the starting material^[1][4].

2.1. Materials Preparation

(1) Reagents: 10% ammonium persulfate solution, 1.5 M Tris-HCl (pH = 8.8), 1.0 M Tris-HCl (pH = 6.8), 10% SDS, 5× electrophoresis buffer stock solution, transfer buffer, TBST buffer, blocking solution (stored at 4°C).
(2) Preparation of protein gel: Assemble the equipment as required and fix it. Seal the bottom of the glass plate with 1% Agarose. Prepare the protein separation gel (3/4 of the height of the flat glass plate) and the protein concentration gel (the height of the flat glass plate). The preparation system is shown in the attached table.

2.2. Methods

2.2.1. Preparation of Cell Extracts
(1) Cell Collection: Collect cells from the culture dish, remove the original culture medium, and wash them with PBS once.
(2) Cell Lysis: Prepare an ice box. Add 500 μL of weak lysis buffer containing 100× protease inhibitor PMSF (HY-B0496) (5 μL) to a 10 cm culture dish for lysing the cell membrane and releasing intracellular proteins. Use a 1 mL pipette to scrape the cells off the culture dish and blow them down with a pipette gun into a 1.5 mL Ep tube (for a 10 cm culture dish, transfer two tubes: experimental group + IgG group). Place the Ep tube in the ice box. At this point, there will be a lot of foam at the tube opening, wait for it to subside. Lysate at 13500 rpm for 15 minutes, then take it out and invert and mix it after a while.
Note: The purpose of using the weak lysis buffer is to retain the natural conformation of proteins, such as the non-ionic denaturant Triton X-100 (HY-Y1883A).
(3) Centrifuge the lysed cells at 13500 rpm for 15 minutes, collect the supernatant (weak lysis product) into a new Ep tube (place in the ice box). Dilute the supernatant with an appropriate buffer 10 times and use the BCA method to quantify the protein concentration. Ensure that the total amount of protein used in each group for the IP experiment is consistent, which is the key to the comparability of the experimental results.
2.2.2. Antibody Incubation
(4) Antibody Binding: Take 450 μL of the weak lysis product for the supernatant, and the remaining 50 μL as the later input control. Add 4 μL of the antibody at 4°C for incubation for 16 hours. The IgG group also takes 450 μL of the supernatant and adds 4 μL of the IgG negative control antibody of the same IgG subclass as the one used.
2.2.3. Binding Medium Incubation
(5) Immunoprecipitation: Prepare solid-phase adsorption substrates (such as Protein A/G magnetic beads (HY-K0202) or agarose beads (coated with protein A/G) (HY-K030). Add 40 μL of agarose beads or 20 μL of magnetic beads to the antigen-antibody complex from step (4), incubate at room temperature for 2 hours (rotator low-speed rotation) (magnetic beads) or at 4°C for overnight incubation (agarose beads).
Note: The specific usage amounts should be in accordance with the product instructions.
Magnetic beads Protein A/G are very prone to agglomeration, so an ultrasonic instrument should be used for assistance. Also, the operation of using a magnetic rack to adsorb magnetic beads should not be operated for a long time, as it will cause the magnetic beads to lose magnetism.
2.2.4. Washing
(6) A. Agarose Bead Washing: After incubation, centrifuge at 2000 rpm for 3 minutes, discard the supernatant, wash with PBS for 5 minutes (wash the beads), centrifuge and discard the supernatant. B. Magnetic Bead Washing: Place the protein-antibody-magnetic bead complex on the magnetic rack, let it stand for 1 minute, discard the supernatant. Add PBS to resuspend the magnetic beads, vortex 15-20 times, let it stand on the magnetic rack for 1 minute, and discard the PBS. Wash 3-5 times.
Note: Agarose bead/magnetic bead washing is to avoid introducing excessive non-specific adsorption of foreign proteins. When carefully aspirating the supernatant, it is better to leave a small amount of supernatant rather than completely removing the agarose bead/magnetic bead. When using agarose beads, the buffer can be removed by using a large pipette tip with a small pipette tip to make the operation more precise.
2.2.5. Elution and Detection
(7) Protein elution: Add 5×loading buffer protein to the Ep tube and heat it in a 100°C water bath for 15 minutes. After centrifugation, take the supernatant for subsequent analysis. At the same time, take the previously reserved Input and perform the operation in step (7).
Note: Since 40 μL of agarose beads were added previously, 8 μL of 5×loading buffer protein should be added.
(8) WB: Take some or all of the samples for Western Blot (WB) experiments to detect the enrichment of proteins. Samples that are not needed for the time being should be stored at -20°C. During WB, the loading volume of the target protein is 20 μL, and the loading volume of Flag is 3 μL.

(1) All operations related to sample processing must be conducted under low-temperature conditions (on ice) and with prevention of freeze-thaw cycles.
(2) For the IP experiment, an Input group (positive control), an IP group, and an IgG group (negative control) need to be set up.
Input group: 5%-10% of the total protein amount
IP group: 1:20 - 1:100 (mass ratio)
IgG group: isotype control of the IP antibody (negative control)
Example: If 200 μg of sample is used for IP, the Input group retains 10-20 μg; the IP group adds 200 μg of sample and 5 μg of IP antibody; the IgG group adds 200 μg of sample and 5 μg of IgG.
(3) As the negative control corresponding to the IP capture antibody experiment group, theoretically, IgG cannot specifically bind to any protein. An isotype IgG from the same species source as the IP capture antibody can be selected as the negative control.
(4) When IgG negative control shows signals other than light and heavy chains, it indicates that there are proteins in the sample that non-specifically bind to IgG. Setting the negative control can be used to exclude non-specific binding of proteins to IgG in the sample, eliminate the background of the sample, and exclude false positives.
(5) IgG, as an independent control group (a control for the IP antibody), needs to undergo exactly the same operations as the experimental group. In simple terms, it is treated as our IP capture antibody for the entire IP experiment process. In the subsequent WB detection, its IP product is analyzed as an independent sample together with the IP product of the experimental group.
(6) Antibody selection follows the recommendation in the antibody selection instruction manual for IP applications. Generally, IP antibodies recognize sites on the three-dimensional structure of proteins, while WB antibodies only need to recognize linear structure sites. If the instruction manual does not indicate that it can be used for IP, antibodies that can be used for immunohistochemistry or immunofluorescence should be selected first. The amount of antibody is determined according to the antibody instruction manual or pre-experimented with a mass ratio (1:50-1:200). The optimal usage amount should be determined through experimentation.
(7) Method to remove the influence of light and heavy chains: Method one: Perform immunoprecipitation and WB experiments using antibodies from different species sources separately, and then select an antibody without cross-reaction between species for WB experiments. Method two: Select special antibodies, such as antibodies that only recognize heavy chains or light chains (anti-Fab and anti-Fc antibodies).
(8) If the detected signal is too weak, it is possible that there is no target protein expression in the sample or the expression level is very low. If the latter is the case, increasing the amount of lysate can be considered, but this will increase non-specific binding. Therefore, it is recommended to pre-purify the lysate before performing the IP.
(9) If the detected signal is too strong, it may be due to poor antibody specificity, using affinity purification and specific antibodies. It may also be due to too much antibody usage causing non-specific binding. Try using less antibodies. Or the protein content in the lysis buffer is too high, resulting in many false positive proteins in the washing solution. Reduce the sample amount.
(10) Experimental conditions can all be optimized, including the amount of agarose beads/magnetic beads, incubation time, washing conditions, etc.

1. RIPA Buffer preparation system

	Reagent Component	Preparation method	Note
a. Basic Component	Tris-HCl		Buffer components to prevent protein denaturation
	NaCl		Salt to prevent non-specific protein aggregation
	NP-40	10%, soluble in H2O	Nonionic detergent for protein extraction
	Sodium Deoxycholate	10%, soluble in H2O	Ionic detergent, for protein extraction
	Note: Do not add sodium deoxycholate to the kinase (activating enzyme) assay. Because ionic detergents can denature enzymes, resulting in loss of activity.
b. RIPA Protease Inhibitor	phenylmethylsulfonyl fluoride (PMSF)	200 mM in isopropanol	Store at room temperature
	EDTA (calcium chelator)	100 mM，dissolved in H2O，pH=7.4
	Leupeptin	1 mg/mL，dissolved in H2O	Aliquot, store at -20°C
	Aprotinin	1 mg/mL，dissolved in H2O	Aliquot, store at -20°C
	Pepstatin	1 mg/mL in methanol	Aliquot, store at -20°C
c. RIPA Phosphatase inhibitors	Na3VO4	200 mM in H2O
	NaF	200 mM in H2O	Store at room temperature
	Note: Do not add phosphatase inhibitors to phosphatase experiments.

2. Protein Glue Preparation System

2-1 SDS-PAGE separation gel configuration system
Reagent Component	Content
ddH2O	3.3 mL
Acryl/Bis30% Solution (29:1)	4.0 mL
1.5M Tris-HCl (pH=8.8)	2.5 mL
10% SDS	100 μL
10% ammonium persulfate	100 μL
TEMED	4 μL
Note: The total volume is enough for two 1.0 mm width protein glue or a piece of 1.5 Protein glue of mm width.

2.2 SDS-PAGE stacking gel configuration system
Reagent components	content
ddH2O	3.4 mL
Acryl/Bis30% Solution (29:1)	830 μL
1.5M Tris-HCl (pH=6.8)	630 μL
10% SDS	50 μL
10% Ammonium persulfate	50 μL
TEMED	5.0 μL
Note: The total volume is enough for two pieces 1.0 mm width of protein glue or a piece 1.5 mm wide protein glue