IP is a method developed by utilizing the specific binding of antigen proteins and antibodies and the phenomenon that “protein A/G” of bacterial proteins specifically binds to FC fragments of antibodies (immunoglobulins). At present, protein A/G is often used to bind to argarose beads in advance, so that after reacting with the solution containing antigen and antibody, 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 the solution containing the antigen of interest.
The immunoprecipitation experiment has many operation steps, and at the same time, because the experiment is carried out under non-denaturing conditions, to obtain a perfect experimental result, not only high-quality antibodies are required, but also strict control indicators for the immunoprecipitation system are required. The immunoprecipitation experiment starts from: protein sample processing; antibody-agarose beads incubation; antibody-agarose beads complex washing to final identification. Each step is very critical. It is necessary to strictly control the quality of each key step in the experimental process in order to finally achieve your goal. the purpose of the experiment.
IP experimental steps
Basic experimental steps
1. Harvest the cells, add an appropriate amount of cell IP lysis buffer (containing protease inhibitors), lyse on ice or at 4°C for 30 min, centrifuge at 12 000g 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 protein A/G-beads to the remaining lysate, and incubate at 4°C with slow shaking overnight;
3. After immunoprecipitation, centrifuge the protein A/G-beads to the bottom of the tube by centrifugation at 3 000 g for 5 min at 4°C; carefully aspirate the supernatant, and use 1 ml of lysis buffer for the protein A/G-beads. Wash 3~4 times; finally add 15 μl of 2×SDS loading buffer, boil with boiling water for 10 minutes;
4. SDS-PAGE, Western blotting or mass spectrometry analysis.
1. Sample processing:
Whether the immunoprecipitation experiment is successful or not, the first step of processing the sample is very critical. An immunoprecipitation experiment is essentially a reaction between an antigen and an antibody in its native conformation state, and the quality of sample processing determines the quality, concentration of the antigen in the antigen-antibody reaction, and whether the antigen is in its native conformation state.
Therefore, the preparation of high-quality samples for subsequent antibody-agarose beads incubation is critical to the success of immunoprecipitation experiments. In this link, in addition to controlling all operations to be done on ice or at 4°, the most critical thing is the composition of the lysate.
The samples used for immunoprecipitation experiments are generally primary culture cell lysates or cell line lysates. We take the commonly used RIPA lysis solution as an example (mainly containing ionic buffer around pH 7.4, NaCl at close to physiological concentration, a certain proportion of detergent and glycerol and various protease inhibitors, etc.) to illustrate its main components , and then help us how to choose the best lysate for different experimental purposes and different protein properties.
1. Buffer: Hepes or Tris-Cl with pH 7.4 is usually used as ionic buffer.
2. The NaCl concentration is generally 150 mM, mainly because 150 mM is close to the physiological concentration and will not destroy the interaction between proteins. However, the intracellular NaCl concentration is not uniform, the local NaCl concentration can be as low as 50 mM, and 150 mM NaCl may disrupt protein interactions in this region. Therefore, the optimal NaCl concentration in the lysate formulation depends on the subcellular localization of the protein being analyzed.
3. Glycerol has a good protective effect on the interaction between proteins due to its viscosity. Typically 10% glycerol is added to help stabilize protein-protein interactions.
4. The detergent in the lysate can lyse the cytoplasmic membrane and also destroy the membranes of many organelles, thereby releasing many of the proteases stored in them. However, since the detergents used in the immunoprecipitation experiments are relatively mild, the activity of the protease is largely preserved. Another part of the protease comes from the cytoplasm, mainly because its inhibitory protein or its activity-inhibiting environment is changed to restore the protease activity. Therefore, the addition of protease inhibitors is very important to prevent the degradation of the target protein to complete the immunoprecipitation experiment. Metalloproteases are generally inhibited mainly by adding EDTA, and protease can be inhibited by Protease Cocktail (a mixture of various protease inhibitors).
5. Detergent is a very critical factor for 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/organelle membrane: Because many target proteins 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, so immunoprecipitation experiments for such proteins need to be cautiously tried with multiple detergents and different concentrations.
(3) Protein interaction: Different detergents have different effects on the interaction of proteins with different properties. It is necessary to analyze and select the type and concentration of detergents according to the characteristics of specific proteins. Since it is difficult to accurately predict which detergents are adapted to act on which proteins, a more practical approach is to screen the appropriate types and concentrations of detergents through specific experiments.
2. Antibody-agarose beads incubation
After lysing cells, centrifuging and removing insoluble membrane components, 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 first and incubated with the sample for several hours, and then protein A or G beads can be added to incubate overnight, or the antibody and Protein A or G beads can be added to incubate overnight.
Generally, 1 mg of total protein (1 mg/ml) is selected to add 1 ug of antibody, and it can be added up to 5 ug of antibody. Too much antibody will cause false positives. The key factor in this step is the selection of an appropriate negative control. Generally, the same amount of IgG is selected, but a more appropriate method is to select a primary antibody against other irrelevant target proteins in the cell as a control.
For example, for 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 cytoplasmic soluble protein C, select another soluble protein D as a negative control control. At the same time, in order to avoid the (non-)specific adsorption of Protein A or G beads, which may cause false positives in the immunoprecipitation test results, generally before adding the target protein antibody, the Protein A or G beads are pre-incubated with the cell lysate for several hours, and then Take the supernatant for subsequent antibody-agarose beads incubation.
At the same time, Protein A or G beads have different affinities for different types of antibodies. In combination with the species and Ig subtype of the primary antibody, choosing 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, which can achieve the best experimental effect and save a lot of trouble of selection.
3. Antibody-agarose beads complex washing:
In addition to selecting antibodies with good specificity and selecting appropriate negative controls, one way to remove non-specificity in immunoprecipitation experiments is to wash the antibody-agarose beads complexes 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. For different experimental requirements, the effect of removing non-specific adsorption can also be achieved by changing the concentration of NaCl and the proportion and type of detergent.
For example, for simple immunoprecipitation rather than co-immunoprecipitation experiments or although co-immunoprecipitation experiments are performed, but the binding between proteins is relatively firm, you can consider using low concentration (0.2-0.5%) SDS to wash the antibody-agarose beads complex , which removes most of the nonspecific interactions.
4. Identification
Immunoprecipitation experiments are very versatile (see IP: Q&A), and many immunoprecipitation-related experimental methods (such as co-immunoprecipitation, chromatin immunoprecipitation, and RNA-protein immunoprecipitation) have been derived from the most basic immunoprecipitation experiments. Therefore, 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 the sample, after the final centrifugation to obtain the antibody-agarose beads complex, the eppendorf tube mainly contains antibody, target protein, Protein A/G beads and some other non-specific hetero-acting protein.
Among them, the antibody, target protein and Protein A/G beads are non-covalently bound together, and only Protein A/G and agarose beads are covalently bound together. Therefore, after adding a loading buffer containing mercaptoethanol, boiling and denaturing and centrifuging out Protein A/G beads, the eppendorf tube contains only antibodies and target proteins and a small amount of non-specifically adsorbed proteins.
In this way, the SDS-PAGE contains two proteins, the target protein and the antibody. Since the loading buffer contains mercaptoethanol, the disulfide bond between the heavy chain and the light chain of the antibody is destroyed, so that the antibody molecule becomes a heavy chain molecule. (55KD) and light chain molecules (25KD). Therefore, in addition to the detection of the target protein in the WB color reaction, heavy and light chain molecules can also be detected if the secondary antibody used belongs to the same genus as the antibody molecule used in the immunoprecipitation experiment.
The amount of antibody usually used for immunoprecipitation is very large (1ug), so when the size of the target protein is close to that of the heavy chain or light chain molecule, the WB signal of the heavy chain or light chain molecule is often too strong to affect the target protein. WB result judgment.
For the above situation, there are usually two solutions:
1. Select antibodies of different species to conduct immunoprecipitation experiments and WB experiments respectively. In this way, selecting a secondary antibody with weak species cross-reaction or no species cross-reaction for WB experiments can greatly weaken the heavy chain and light chain molecules. WB signal.
2. Use a cross-linking agent to cross-link the antibody and Protein A/G beads, then process the target protein-antibody-agarose beads complex by adding loading buffer without mercaptoethanol, and finally remove the antibody-agarose beads complex by centrifugation. Only the target protein is left in the supernatant.
