IP is a method developed by the specific binding of an antigenic protein and an antibody and the phenomenon that a "protein A/G" of a bacterial protein specifically binds to an FC fragment of an antibody (immunoglobulin). At present, protein A/G is pre-bound on argarose beads to react with the antigen-containing solution and antibody, and the prorein A/G on the beads can achieve the purpose of adsorbing antigen. By low-speed centrifugation, the antigen of interest can be separated from other antigens from a solution containing the antigen of interest.
Immunoprecipitation experiments have many operational steps, and because of the experiments under non-denaturing conditions, a perfect experimental result is required, not only high-quality antibodies, but also strict control indicators for the immunoprecipitation system. Immunoprecipitation experiments from: protein sample treatment; antibody-agarose beads incubation; antibody-agarose beads complex wash to final identification, each step is very critical, the quality of each critical step in the experimental process needs to be strictly controlled, in order to finally reach you Experimental purpose.
IP experiment steps
Basic experimental steps
1. Harvest the cells, add appropriate amount of cell IP lysis buffer (including protease inhibitor), cleave on ice or at 4 ° C for 30 min, centrifuge at 12 000 g for 30 min, and take the supernatant;
2. Take a small amount of lysate for Western blot analysis. Add 1 μg of the corresponding antibody and 10~50 μl of protein A/G-beads to the cell lysate for the remaining lysate, and incubate slowly at 4 °C overnight.
3. After immunoprecipitation, centrifuge at 3 000 g for 5 min at 4 ° C, centrifuge protein A/G-beads to the bottom of the tube; carefully aspirate the supernatant, and use protein 1/G-beads with 1 ml lysis buffer. Wash 3~4 times; finally add 15 μl of 2×SDS loading buffer and boil for 10 minutes.
4. SDS-PAGE, Western blotting or mass spectrometry.
First, sample processing:
Whether the immunoprecipitation experiment is successful or not, it is very important to process the sample in the first step. The immunoprecipitation experiment is essentially a reaction between an antigen and an antibody in a native conformational state, and the quality of the sample treatment determines the quality, concentration, and whether the antigen is in a native conformational state in the antigen-antibody reaction.
Therefore, the preparation of high quality samples for subsequent antibody-agarose beads incubation is critical to the success of immunoprecipitation experiments. In this step, in addition to controlling all operations as much as possible on ice or 4°, the most critical component of the lysate.
Samples used in immunoprecipitation experiments are typically primary cultured cell lysates or cell line lysates. We use the commonly used RIPA lysate as an example (mainly containing an ion buffer of about pH 7.4, close to NaCl at physiological concentration, a certain proportion of detergent and glycerol, and various protease inhibitors) to explain the main components. Its use, in turn, helps us choose the best lysate for different experimental purposes and different protein properties.
1. Buffer: Ion buffer is often used with Hepes or Tris-Cl at pH 7.4.
2. NaCl concentration is generally used to 150 mM, mainly because 150 mM is close to physiological concentration and does not destroy the interaction between proteins. However, the NaCl concentration inside the cells is not uniform, and the local NaCl concentration can be as low as 50 mM. 150 mM NaCl may damage the protein interaction in this region. Therefore, the optimum NaCl concentration for the lysate formulation depends on the subcellular localization of the protein being analyzed.
3. Glycerin, because of its viscosity, can play a good role in protecting the interaction between proteins. The addition of 10% glycerol generally helps to stabilize the interaction between proteins.
4. The detergent in the lysate cleaves the plasma membrane and also destroys many of the organelle membranes, releasing many of the proteases stored therein. Since the detergent used in the immunoprecipitation experiment is mild, most of the protease activity is preserved. A further portion of the protease is derived from the cytosol, and the protease activity is restored mainly due to changes in its inhibitory protein or its activity inhibiting environment. Therefore, the addition of protease inhibitors is critical to the prevention of degradation of the protein of interest to complete immunoprecipitation experiments. Protease is inhibited primarily by the addition of EDTA to inhibit metalloproteinases and by Protease Cocktail (a mixture of protease inhibitors).
5. Detergents are a critical factor in immunoprecipitation experiments, especially co-immunoprecipitation experiments. Different detergent types and different detergent concentrations affect the immunoprecipitation effect mainly by affecting the following three factors:
(1) Permeability of cytoplasm/membrane: Since many proteins of interest are localized in organelles, these proteins must be released before antibodies can react with them.
(2) Release of membrane proteins: The conformation of many membrane proteins is very sensitive to the type and concentration of detergents. Therefore, immunoprecipitation experiments for such proteins require careful trials of various detergents and different concentrations.
(3) Protein interaction: Different detergents have different effects on the interaction of proteins of different natures, and it is necessary to analyze the type and concentration of detergent according to the characteristics of specific proteins. And because of which detergents are adapted to which proteins are difficult to predict accurately, a more practical approach is to screen for appropriate detergent types and concentrations through specific experiments.
Second, antibody-agarose beads incubation
After lysing the cells, centrifuging and removing the insoluble membrane fraction, the supernatant can be stored at -80° for 3 months, but it is best to use freshly prepared cell lysate supernatant for antibody-agarose beads incubation experiments. The antibody can be added to the supernatant for several hours after incubation with the sample and then incubated with Protein A or G beads overnight. Alternatively, the antibody can be incubated with Protein A or G beads overnight.
Generally, 1 mg of total protein (1 mg/ml) is added to add 1 ug of antibody, up to 5 ug of antibody, and too many antibodies produce false positives. The key factor in this step is the selection of a suitable negative control. It is generally preferred to add the same amount of IgG, but a more appropriate method is to select a primary antibody against other intracellular proteins of interest.
For example, to perform membrane immunoprecipitation of membrane protein A, select membrane protein B as a negative control, as long as there is no interaction between the two; and for immunoprecipitation of cytosolic soluble protein C, select another soluble protein D to be negative. Control. At the same time, in order to avoid the (non)specific adsorption of Protein A or G beads, which may cause false positive results in immunoprecipitation experiments, Protein A or G beads are usually incubated with cell lysate for several hours before adding the antibody of the target protein, and then The supernatant was taken for subsequent incubation with antibody-agarose beads.
At the same time, Protein A or G beads have different affinity for different types of antibodies. Combined with the species of the primary antibody and the Ig subtype, selecting the appropriate Protein A or G beads is also an important factor in determining the success of the immunoprecipitation experiment. It is generally recommended to use a mixture of Protein A and Protein G beads to achieve the best results and eliminate the hassles of many choices.
Third, antibody-agarose beads complex washing:
In addition to selecting specific antibodies and selecting a suitable negative control, one way to remove non-specific immunoprecipitation experiments is to wash the antibody-agarose beads complex multiple times.
The general wash buffer uses the same formulation as the lysate, but removes glycerol to reduce non-specific adsorption due to the viscosity of glycerol. According to different experimental requirements, the effect of removing non-specific adsorption can also be achieved by changing the concentration of NaCl and the proportion of detergent.
For example, for simple immunoprecipitation rather than co-immunoprecipitation experiments or although co-immunoprecipitation experiments, but the binding between proteins is relatively robust, it can be considered to use low concentration (0.2-0.5%) of SDS to wash antibody-agarose beads complex This allows removal of most non-specific interactions.
Fourth, identification
Immunoprecipitation experiments are very versatile (see IP: Q&A), and many immunoprecipitation-related assays (such as co-immunoprecipitation, chromatin immunoprecipitation, and RNA-protein immunoprecipitation) have been derived based on the most basic immunoprecipitation experiments. The identification method of the immunoprecipitation experiment mainly depends on the purpose of the experiment.
Since the immunoprecipitation experiment uses the target protein antibody plus Protein A/G beads to incubate with the sample, after the final centrifugation to obtain the antibody-agarose beads complex, the eppendorf tube mainly contains antibodies, protein of interest, Protein A/G beads and some other non-special Heterologous protein.
Among them, the antibody and the target protein and Protein A/G beads are combined with non-covalent bonds, and only Protein A/G and agarose beads are covalently bound together. Therefore, after the addition of the thioglycol-containing loading buffer and the boiling denaturation and centrifugation of the Protein A/G beads, the eppendorf tube has only the antibody and the target protein and a small amount of non-specific adsorbed protein.
Thus, SDS-PAGE contains the target protein and the antibody, and the disulfide bond between the heavy chain and the light chain of the antibody is destroyed due to the presence of mercaptoethanol in the loading buffer, thereby causing the antibody molecule to become a heavy chain molecule. (55KD) and light chain molecules (25KD). Therefore, in addition to the detection of the protein of interest in the WB color reaction, if the secondary antibody used is of the same genus as the antibody molecule used in the immunoprecipitation experiment, heavy and light chain molecules can also be detected.
The amount of antibody commonly used for immunoprecipitation is very large (1 ug), so when the size of the target protein is close to that of a heavy or light chain molecule, the WB signal of a heavy or light chain molecule often affects the target protein due to excessive signal. WB results judged.
In response to the above, there are usually two solutions:
1. Select antibodies of different species for immunoprecipitation and WB experiments, and then select a secondary antibody with weak cross-reaction or cross-reaction with no species to carry out WB experiments, which can greatly weaken heavy and light chain molecules. WB signal.
2. Crosslink the antibody with Protein A/G beads using a cross-linking agent, then treat the protein-antibody-agarose beads complex by adding a buffer solution containing no mercaptoethanol, and finally remove the antibody-agarose beads complex by centrifugation. Only the protein of interest is left in the supernatant.
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