Immunoprecipitation Protocol for Native Proteins
Licia Miller Product Manager
Immunoprecipitation of natural proteins is a classic method for studying natural protein interactions, which relies on the specific binding between antibodies and proteins (antigens). This technique can be used to qualitatively detect proteins, determine the physiological role of proteins in cells, and discover new binding proteins.
This protocol describes the analysis of native proteins using western blotting.
1. Preparation of Cell Lysates
Required solutions and reagents
Note: Prepare solutions using reverse osmosis deionized (RODI) or equivalent grade water.
(1) 1 L 20× Phosphate Buffered Saline (PBS) :
50 ml 20× PBS to 950 ml dH2O and mix thoroughly.
(2) 10 ml 10× cell lysis buffer:
1 ml of 10× cell lysis buffer to 9 ml of dH2O and mix thoroughly. Note that 1 mM PMSF needs to be added before use.
(3) 3× SDS sample buffer:
Prepare fresh 3× reducing loading buffer by adding 1/10 volume of 30× DTT to 1 volume of 3× SDS loading buffer.
Experimental Steps
1. Aspirate media. Treat cells by adding fresh media containing regulator for desired time.
2. Remove the culture medium and wash the cells once with ice-cold 1× PBS.
3. Remove PBS and add 0.5 ml of ice-cold 1× cell lysis buffer to each 10 cm cell plate and incubate on ice for 5 minutes.
4. Scrape the cells from the plate and transfer to a microcentrifuge tube and place on ice.
5. Sonicate on ice 3 times for 5 seconds each.
6. Microcentrifuge at 14,000 × g for 10 minutes at 4°C.
7. Transfer the supernatant to a new tube. The supernatant is the cell lysate. If no subsequent operations are performed, the lysate can be stored at -80°C.
2. Cell Lysate Pre-clearing (Optional)
Required solutions and reagents
(1) Protein A or G agarose beads (for unconjugated primary antibodies):
Rabbit IgG immunoprecipitation and mouse IgG immunoprecipitation were performed using Protein A and Protein G, respectively.
(2) Immobilized streptavidin (coupled to beads) (for biotinylated antibodies):
Vortex the vial gently and use 10 µl in each immunoprecipitation.
Experimental Steps
Note : It is highly recommended to use an isotype control antibody to distinguish specific recognition from non-specific binding. Isotype controls should be concentration matched and run simultaneously with the primary antibody samples.
1. Add 20 μl of Protein A or Protein G agarose 50% bead slurry and 10 μl Streptavidin (UltraBio™ Streptavidin Magnetic Beads) to 200 μl of cell lysate at a concentration of 1 mg/ml for biotinylated antibody detection. Incubate with rotation at 4°C for 30-60 minutes.
2. Microcentrifuge at 4°C for 10 minutes.
3. Transfer the supernatant to a new tube and proceed with the following experimental steps according to the primary antibody used.
3. Antibody Incubation
● For unconjugated primary antibodies
1. Add primary antibody (at the appropriate dilution recommended in the product datasheet) to 200 µl of cell lysate. Incubate overnight at 4°C with gentle shaking.
2. Add Protein A or Protein G agarose (20 µl of 50% bead slurry). Shake gently and incubate at 4°C for 1-3 hours.
3. Microcentrifuge at 4°C for 30 seconds.
4. Wash the pellet five times with 500 µl of 1× cell lysis buffer. Keep the samples on ice between washes.
● For biotin conjugated primary antibodies
1. Add biotin conjugated antibody (at the appropriate dilution recommended in the product datasheet) to 200 µl of cell lysate. Incubate overnight at 4°C with gentle shaking.
2. Gently mix the immobilized Streptavidin (UltraBio™ Streptavidin Magnetic Beads) and add 10 µl of bead slurry. Incubate at 4°C for 2 hours with gentle shaking.
3. Microcentrifuge at 4°C for 30 seconds.
4. Wash the pellet five times with 500 µl of 1× cell lysis buffer. Keep samples on ice between washes.
4. Sample Analysis by Western Blotting
Note: Do not centrifuge the beads. Use a magnetic separation stand instead.
1. Resuspend the pellet in 20-40 µl 3× SDS sample buffer. Vortex and microcentrifuge for 30 seconds.
2. Heat the sample to 95-100°C for 2-5 minutes and then centrifuge briefly at 14,000 × g for 1 minute.
3. Load 15-30 µl of sample onto an SDS-PAGE gel. Analyze sample by western blotting (see General Protocol for Western Blotting).
For more product details, please visit Aladdin Scientific website.