實驗概要
Immunoprecipitation is a procedure by which proteins or peptides that react specifically with an antibody are removed from solution and examined for quantity or physical characteristics. Immunoprecipitation can also be used to “enrich” a protein population prior to Western blotting. For example, immunoprecipitation with a pan-specific antibody against a protein of interest followed by Western blotting with a modification-specific antibody (such as a phosphospecific antibody or an acetylation-specific antibody).
主要試劑
1X Cell Lysis Buffer:
20 mM Tris-HCl, pH 7.5
1 μg/ml aprotinin
150 mM NaCl
1 mM Na3PO4
1% NP-40
1 mM PMSF
2 mM EDTA
5 mM NaF
1 μg/ml leupeptin
3 mM Na4P2O4
Protein A/G sepharose:
Transfer 30 μl protein A or G sepharose per sample in a microcentrifuge tube, wash twice in cell lysis buffer to remove preservative (add cell lysis buffer up to 800 μl and spin down in benchtop centrifuge). Resuspend protein A or G sepharose in cell lysis buffer at 100 μl per sample.
5X SDS sample Buffer:
312.5 mM Tris-HCl (pH 6.8)
10% SDS (w/v)
250 mM DTT
50% Glycerol
0.05% Bromophenol Blue (w/v) Use at 1X
10X SDS Running Buffer:
Dissolve 144 g of Glycine, 30 g of Tris base and 10 g SDS in 800 ml of distilled H2O.
Add distilled H2O to 1 liter
Use at 1X
Transfer Buffer:
2.25 g Tris base
10.5 g Glycine
1 g SDS
200 ml Methanol
Add distilled water to 1.0 L
實驗步驟
1. Sample preparation:
1) Collect cells and transfer to a microcentrifuge tube and centrifuge at 3000 rpm for 5 min at 4°C.
2) Discard the supernatant and store the pellet at -80°C or process immediately as described below.
2. Immunoprecipitation:
1) Remove samples from -80°C freezer and place on ice.
2) Immediately add 800 μl ice-cold lysis buffer to the samples and vortex, then incubate for 30 min on ice.
3) Spin lysates at 14,000 rpm in a pre-cooled centrifuge for 10 min.
4) Transfer 720 μl supernatant to a fresh pre-cooled microcentrifuge tube containing washed protein A or G agarose bead slurry (30 μl packed beads) and rock the mixture for 30 min at 4°C. Collect the agarose beads by pulsing in a microcentrifuge (5 seconds at 14,000 rpm, 4°C).
5) Remove supernatant and place in a fresh pre-cooled microcentrifuge tube containing ~4 μg specific antibody and rock gently at 4°C for 2 hrs. Capture the immune complex by adding 100 μl of washed protein A or G agarose bead slurry (30 μl packed beads) and rock the mixture for 1 hr at 4°C.
6) Collect the agarose beads by pulsing in a microcentrifuge (5 seconds at 14,000 rpm, 4°C). Aspirate and discard supernatant. Wash the beads 3 times with ice-cold cell lysis buffer.
7) After final wash, remove supernatant and add 40 μl 2X SDS sample buffer. Boil for 5 min at 95°C and spin down the beads at maximum speed in a microcentrifuge for 5 min at room temperature. Carefully pipette off supernatant.
8) Load up 30 μl of sample in each well of a 1.5 mm thick gel. Run gel according to manufacturer’s recommendations and continue with immunoblotting using BioLegend’s Western Blotting protocol, p. 31 (alternately, radio labeled proteins prepared from target cells can be used to directly visualize the immunoprecipitated protein).
注意事項
1. The choice of lysis buffer depends on the location of the protein (membrane, cytosolic, nuclear).
2. Immunoprecipitates allowed to incubate overnight may have a higher background than ones processed for shorter periods of time due to time- dependent aggregation or denaturation of cellular proteins.
3. Always use an isotype-matched irrelevant control antibody (monoclonal) or same-species serum from a non-immunized animal to remove non-specific antibody binding in cellular lysates.
