- Western blotting guide
- Western blotting buffers & recipes
- Western blot sample protocol
- Western blotting dictionary
- Western blot troubleshooting tips
- Mammalian cell & tissue culture guide
- How to use a haemocytometer
- RIPA Buffer – Cell lysis buffer
- Subcellular fractionation protocol
Co-immunoprecipitation (co-IP) sample protocol
Co-immunoprecipitation is carried out in order to isolate a target protein and it’s binding partners from whole cell lysates. The lysate is incubated with a protein-specific antibody. The antibody/antigen complex is pulled out of the sample using protein A/G-coupled agarose beads, which isolate your protein of interest. The protein of interest is then separated from the agarose beads by centrifugation and analysed by Western blot. Below you will find everything you need to carry out co-immunoprecipation (co-IP), buffers and solutions, an optimized protocol and hint and tips for the perfect IP.
Buffers and Solutions
IP Lysis Buffer
- 50 mM HEPES, pH 7.5,
- 150 mM NaCl,
- 1 mM EDTA,
- 5 mM EGTA,
- 1% (w/v) Tween 20,
- 1 mM dithiothreitol,
- 1 mM NaF
- 100 µM PMSF
- 1% v/v NP-40
- 20mM Tris-HCL pH 7.4
- 5mM Sodium Pyrophosphate
- 5mM EDTA
- Freshly added proteinase Inhibitors (Leupeptin, PMSF, Sodium Ortovanadate)
Co-immunoprecipitation (co-IP) protocol for the isolation of proteins from whole cell extracts
- Harvest cells by centrifugation at 400 x g for 3 min.
- Aspirate the media.
- Resuspend the cells in 500 µl of IP lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 0.1% (w/v) Tween20, 1 mM dithiothreitol, 1 mM NaF and 100 µM PMSF) or RIPA buffer and leave on ice for 15 min.
- Sonicate cells for 2 x 10 sec and place directly on ice.
- Centrifuge at 10,000 x g for 10 min in an at 4 °C.
- Transfer the supernatant to a fresh cold Eppendorf.
- Determine the protein concentration by Bradford assay (see below).
- Pre-clear whole cell extracts by adding 1-2 mg of protein to a fresh Eppendorf containing 20 µl of A/G sepharose beads and 2 µg of rabbit or mouse IgG control antibody.
- Rotate samples for 1 h at 4 °C.
- Centrifuge at 1000 x g for 1 min at 4 °C to pellet the IgG bound-A/G sepharose beads.
- Discard the bead pellet and keep supernatant for immunoprecipitation.
- Transfer the supernatant to a fresh Eppendorf tube containing either rabbit or mouse IgG control antibody (2 µg) or the relevant antibody (2 µg) plus A/G sepharose beads (40 µl) or A/G sepharose beads only.
- Make up the sample volume to 500 µl using IP lysis buffer.
- Rotate samples overnight at 4 °C. To collect the immunoprecipitated protein.
- Centrifuge samples at 1000 x g for 1 min at 4 °C to pellet the antibody-bound A/G sepharose beads.
- Aspirate the supernatant.
- Resuspend the beads in 1 ml of IP lysis buffer or RIPA buffer and centrifuge at 1000 x g for 1 min at 4 °C.
- Perform step 6 x 3 times to wash the beads of non-specific proteins.
- Resuspend the beads in 6X Laemmli buffer
- Boil for 1 min at 100 °C,
- Analyse immediately by SDS-PAGE and Western blotting or stored at -20 °C until analysis.
Helpful hints for co-immunoprecipitation
The quality of the lysate you use for co-immunoprecipitation will determine the success of your assay. Using the right lysis buffer can greatly improve the quality of your lysate. An ideal lysis buffer should stabilize the native protein conformation, inhibit enzymatic activity, prevent denaturation and above all ensure maximum release of proteins from cells or tissues. Non-ionic detergents like NP-40 and Triton X-100 are less harsh when compared to ionic detergents such as SDS and sodium deoxycholate. The use of denaturing buffers such as radio-immunoprecipatation (RIPA) are ideal for proteins that are difficult to release such as nuclear proteins.
Detergent-free buffers can also be used if the target protein can be released from cells by physical disruption, such as mechanical homogenization or heat. Always remember that proteolysis, de-phosphorylation and denaturation can start as soon as cell lysis occurs. This can be slowed down by keeping the samples on ice or at 4°C at all times and through the addition of protease and phosphatase inhibitors to the lysis buffer.
Preclearing the lysates
Preclearing the lysates with the beaded support before beginning co-immunoprecipitation helps to remove any potentially reactive components which may bind non-specifically to the bead components. This preclearing step can also be performed using a non-specific antibody of the same species of origin and isotype as the capture antibody. This process will remove anything that might bind non-specifically to the capture antibody during co-immunoprecipitation. The end result will be a lowering of background and an improved signal-to-noise ratio.
Choosing the right antibody for purification is critical as it can alter your protein yield. Polyclonal antibodies are ideal for the binding of your target protein as they can bind multiple epitopes on the target protein, and form tighter binding immune-complexes with higher retention rates. A combination of a polyclonal capture antibody and a monoclonal antibody for detection will guarantee maximum capture efficacy with high detection specificity. It should also be noted that the use of secondary antibodies which recognize the heavy and light-chain of the primary antibody for western blot detection of IP samples will always result in two bands (the heavy-chain at 50kDa and the light-chain at 25kDa).
Wash buffer Choice
The wash buffer used for co-immunoprecipitation assays should reduce non-specific protein binding and maintain desired protein interactions. PBS and TBS are commonly used as wash buffers as they have physiological concentrations of salt and pH. Moderate adjustments to the salt concentration of wash buffers can be used to reduce background in some instances.
If non-specific interactions are detected using the above wash buffers the stringency may be increased by increasing the sodium chloride concentration. A low level of reducing agents (such as 1-2 mM DTT or β-mercaptoethanol) can help disrupt non-specific interactions.
Elution Buffer Choice
The strength and pH of your elution buffer ensures the correct elution of your target protein. If the immunoprecipitated sample is going to be further analysed by SDS-PAGE or Western Blot elution into running buffer would be ideal. Elution in a milder buffer (0.1 M glycine, pH 2.5) and neutralizing before loading to SDS-PAGE gel is also an option.
Bradford Assay for protein determination
- Aliquot (2 ml) of each protein sample was into separate wells of a 96 well plate.
- Add Bradford reagent (100 ml) to each sample. Ensure that no bubbles are introduced.
- Gently shake at room temperature for 5 min.
- To calculate the protein concentration in each sample read the absorbance off a BSA standard curve, constructed as follows: prepare serial dilutions of BSA between 2 mg/ ml and 15 mg/ml and add to 100 ml of Bradford reagent in a 96 well plate.
- Measure absorbance at 595 nm, normalise to a reference measurement at 450 nm against the blank (2 ml lysis buffer, 100 ml dye reagent) on a Pro-Max5 microplate reader.
- The protein sample concentration can be calculated against the standard curve.