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2023-07-12 23:13| 来源: 网络整理| 查看: 265

Immunoprecipitation for Native Proteins

This protocol is intended for immunoprecipitation of native proteins for analysis by western immunoblot or kinase activity utilizing Protein A magnetic separation.

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L of 1X PBS, add 50 ml 20X PBS to 950 ml dH2O, mix.

10X Cell Lysis Buffer: (#9803) To prepare 10 ml of 1X cell lysis buffer, add 1 ml cell lysis buffer to 9 ml dH2O, mix.

NOTE: Add 1 mM PMSF (#8553) immediately prior to use.

3X SDS Sample Buffer: Blue Loading Pack (#7722) or Red Loading Pack (#7723) Prepare fresh 3X reducing loading buffer by adding 1/10 volume 30X DTT to 1 volume of 3X SDS loading buffer. Protein A Magnetic Beads: (#73778). Magnetic Separation Rack: (#7017) or (#14654). 10X Kinase Buffer (for kinase assays): (#9802) To Prepare 1 ml of 1X kinase buffer, add 100 µl 10X kinase buffer to 900 µl dH2O, mix. ATP (10 mM) (for kinase assays): (#9804) To prepare 0.5 ml of ATP (200 µM), add 10 µl ATP (10 mM) to 490 µl 1X kinase buffer. B. Preparing Cell Lysates Aspirate media. Treat cells by adding fresh media containing regulator for desired time. To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold 1X PBS. Remove PBS and add 0.5 ml ice-cold 1X cell lysis buffer to each plate (10 cm) and incubate on ice for 5 min. Scrape cells off the plate and transfer to microcentrifuge tubes. Keep on ice. Sonicate on ice three times for 5 sec each. Microcentrifuge for 10 min at 4°C, 14,000 x g and transfer the supernatant to a new tube. The supernatant is the cell lysate. If necessary, lysate can be stored at -80°C. C. Immunoprecipitation Cell Lysate Pre-Clearing (Optional)

A cell lysate pre-clearing step is highly recommended to reduce non-specific protein binding to the Protein A Magnetic beads. Pre-clear enough lysate for test samples and isotype controls.

Briefly vortex the stock tube to resuspend the magnetic beads.

IMPORTANT: Pre-wash #73778 magnetic beads just prior to use:

Transfer 20 μl of bead slurry to a clean tube. Place the tube in a magnetic separation rack for 10-15 seconds.

Carefully remove the buffer once the solution is clear. Add 500 μl of 1X cell lysis buffer to the magnetic bead pellet, briefly vortex to wash the beads. Place tube back in magnetic separation rack. Remove buffer once solution is clear. Repeat washing step once more.

Add 200 μl cell lysate to 20 μl of pre-washed magnetic beads.

IMPORTANT: The optimal lysate concentration will depend on the expression level of the protein of interest. A starting concentration between 250 μg/ml-1.0 mg/ml is recommended.

Incubate with rotation for 20 minutes at room temperature. Separate the beads from the lysate using a magnetic separation rack, transfer the pre-cleared lysate to a clean tube, and discard the magnetic bead pellet. Proceed to immunoprecipitation section. Immunoprecipitation

IMPORTANT: Appropriate isotype controls are highly recommended in order to show specific binding in your primary antibody immunoprecipitation. Use Normal Rabbit IgG #2729 for rabbit polyclonal primary antibodies, Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 for rabbit monoclonal primary antibodies, Mouse (G3A1) mAb IgG1 Isotype Control #5415 for mouse monoclonal IgG1 primary antibodies, Mouse (E5Y6Q) mAb IgG2a Isotype Control #61656 for mouse monoclonal IgG2a primary antibodies, Mouse (E7Q5L) mAb IgG2b Isotype Control #53484 for mouse monoclonal IgG2b primary antibodies, and Mouse (E1D5H) mAb IgG3 Isotype Control #37988 for mouse monoclonal IgG3 primary antibodies. Isotype controls should be concentration matched and run alongside the primary antibody samples.

Add primary antibody (at the appropriate dilution as recommended in the product datasheet) to 200 µl cell lysate. Incubate with rotation overnight at 4°C. to form the immunocomplex. Pre-wash magnetic beads (see Cell Lysate Pre-Clearing section, steps 1 and 2). Transfer the lysate and antibody (immunocomplex) solution to the tube containing the pre-washed magnetic bead pellet. Incubate with rotation for 20 min at room temperature. Pellet beads using magnetic separation rack. Wash pellets five times with 500 μl of 1X cell lysis buffer. Keep on ice between washes. Proceed to analyze by western immunoblotting or kinase activity (section D). D. Sample Analysis

Proceed to one of the following specific set of steps.

For Analysis by Western Immunoblotting Resuspend the pellet with 20-40 µl 3X SDS sample buffer, briefly vortex to mix, and briefly microcentrifuge to pellet the sample. Heat the sample to 95-100°C for 5 min. Pellet beads using magnetic separation rack. Transfer the supernatant to a new tube. The supernatant is the sample. Analyze sample by western blot (see Western Immunoblotting Protocol).

NOTE: To minimize masking caused by denatured IgG heavy chains (~50 kDa), we recommend using Mouse Anti-Rabbit IgG (Light-Chain Specific) (D4W3E) mAb (#45262) or Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127). To minimize masking caused by denatured IgG light chains (~25 kDa), we recommend using Mouse Anti-Rabbit IgG (Conformation Specific) (L27A9) mAb (#3678) (or HRP conjugate #5127).

For Analysis by Kinase Assay Wash pellet twice with 500 µl 1X kinase buffer. Keep on ice. Suspend pellet in 40 µl 1X kinase buffer supplemented with 200 µM ATP and appropriate substrate. Incubate for 30 min at 30°C. Terminate reaction with 20 µl 3X SDS sample buffer. Vortex, then microcentrifuge for 30 sec. Transfer supernatant containing phosphorylated substrate to another tube. Heat the sample to 95-100°C for 2-5 min and microcentrifuge for 1 min at 14,000 x g. Load the sample (15-30 µl) on SDS-PAGE gel.

posted December 2008

revised April 2021

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