Immobilized IP Protocol
Licia Miller Product Manager
Immunoprecipitation (IP) experiments are important techniques in biochemistry and molecular biology for studying protein interactions and protein expression status. Depending on the specific needs and conditions of the experiment, different IP methods can be used, mainly including magnetic bead IP and immobilized IP.
Magnetic bead IP uses protein A or protein G magnetic beads combined with specific antibodies to capture the target protein. This method is easy to operate, fast, and has high separation efficiency, making it particularly suitable for high-throughput screening and automated experiments. The magnetic beads are quickly separated by a magnetic separation rack, making the experimental steps more concise. In addition, magnetic bead IP is particularly useful in studying protein-protein interactions and signal transduction pathways.
Immobilized IP uses an immobilized antibody (usually an antibody coupled to beads) to capture the target protein and is commonly used for sample preparation prior to Western Blot analysis. This method requires longer incubation times to form immune complexes, but provides highly specific and sensitive results and is suitable for quantitative analysis of the expression and modification status of the target protein. Immobilized IP is particularly important when studying protein expression levels, post-translational modifications, or protein stability.
The specific operation of magnetic bead IP method has been introduced before. This article will describe the experimental operation of immobilized IP method in detail.
Required solutions and reagents
(1) PBS buffer.
(2) 1× Cell Lysis Buffer: 20 mM Tris (pH 7.5) , 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM Na3VO4, 1 µg/ml leupeptin.
(3) 3× SDS sample buffer: 187.5 mM Tris-HCl (pH 6.8, 25°C), 6% w/v SDS, 30% glycerol, 150 mM DTT, 0.03% w/v bromophenol blue.
Note: All the above solutions were prepared with ultrapure water.
Cell lysate was supplemented with 1 mM PMSF before use.
1. Preparation of Cell Lysates
1.1 Aspirate the culture medium. Add fresh culture medium containing regulatory factors and treat the cells for a period of time according to the experimental purpose.
1.2 Remove the culture medium and wash the cells once with ice-cold PBS.
1.3 Remove PBS and add 0.5 ml of pre-cooled 1× cell lysis buffer and 1 mM PMSF to each cell culture plate (10 cm).
1.4 Incubate on ice for 5 minutes.
1.5 Gently scrape the cells from the plate, transfer to a microcentrifuge tube and place on ice.
1.6 Sonicate the sample on ice four times for 5 seconds each.
1.7 Microcentrifuge at 4°C for 10 minutes and transfer the supernatant to a new tube.
1.8 Proceed to the next step as soon as possible. If you are not going to perform subsequent experiments, store the lysate at -80°C.
2. Immunoprecipitation
2.1 Take 200 μl of cell lysate and add 10 μl of immobilized antibody, place on a shaker and gently shake, and incubate overnight at 4°C.
2.2 Microcentrifuge at 4°C for 30 seconds.
2.3 Resuspend the pellet in 500 µl of 1× cell lysis buffer and then centrifuge to remove the supernatant. Wash five times in total. Keep the samples on ice between washes.
2.4 Resuspend the pellet in 20 μl 3× SDS sample buffer. Vortex and then microcentrifuge for 30 seconds.
2.5 Heat the sample to 95-100°C for 2-5 minutes.
2.6 Load 15-30 µl of the sample onto an SDS-PAGE gel and perform western blot (see Western Blotting Protocol).
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