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Co-Immunoprecipitation (co-IP) Protocol

What is co-IP?

Co-immunoprecipitation is a popular tool that 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.

Co-IP lysis buffers

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

RIPA Buffer

  • 1% v/v NP-40
  • 20mM Tris-HCL pH 7.4
  • 5mM Sodium Pyrophosphate
  • 5mM EDTA
  • Freshly added proteinase Inhibitors (Leupeptin, PMSF, Sodium Ortovanadate)

Protein A & G agarose beads

Protein G Agarose Beads are an affinity matrix for the small-scale isolation of immunocomplexes from immunoprecipitations (IP assays). Protein G is covalently coupled to agarose beads, which enables it to bind to various antibodies, including those of the IgG class. This makes protein G agarose beads an ideal tool for immunoprecipitation experiments involving protein A and G antibodies.

Protein G agarose beads can be used in a variety of immunoprecipitation assays, including those involving protein A and G antibodies.

Some of the advantages of using protein G agarose beads include:

  • They are easy to use and can be stored at room temperature.
  • Protein G agarose beads have a high binding capacity for antibodies, which makes them ideal for small-scale immunoprecipitation experiments.
  • Protein G agarose beads can be used with a variety of antibodies, including those of the IgG class.
  • Protein G agarose beads are a versatile tool for immunoprecipitation experiments involving protein A and G antibodies.

Co-IP protocol (Co-immunoprecipitation) for the isolation of proteins from whole cell extracts

Steps Procedure

1.

Harvest cells by centrifugation at 400 x g for 3 min.

2.

Aspirate the media.

3.

Re-suspend 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.

4.

Sonicate cells for 2 x 10 sec and place directly on ice.

5.

Centrifuge at 10,000 x g for 10 min in an at 4 °C.

6.

Transfer the supernatant to a fresh cold Eppendorf.

7.

Determine the protein concentration by Bradford assay (see below).

8.

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.

9.

Rotate samples for 1 h at 4 °C.

10.

Centrifuge at 1000 x g for 1 min at 4 °C to pellet the IgG bound-A/G sepharose beads.

11.

Discard the bead pellet and keep supernatant for immunoprecipitation.

12.

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.

13.

Make up the sample volume to 500 µl using IP lysis buffer.

14.

Rotate samples overnight at 4 °C. To collect the immunoprecipitated protein.

15.

Centrifuge samples at 1000 x g for 1 min at 4 °C to pellet the antibody-bound A/G sepharose beads.

16.

Aspirate the supernatant.

17.

Re-suspend the beads in 1 ml of IP lysis buffer or RIPA buffer and centrifuge at 1000 x g for 1 min at 4 °C.

18.

Perform step 6 x 3 times to wash the beads of non-specific proteins.

19.

Resuspend the beads in 6X Laemmli buffer

20.

Boil for 1 min at 100 °C

21.

Analyse immediately by SDS-PAGE and Western blotting or stored at -20 °C until analysis.


Helpful Tips for Co-Immunoprecipitation

Lysate Preparation

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.

Pre-clearing the lysates

Pre-clearing 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 pre-clearing 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.

Antibody Choice

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

Steps Procedure

1.

Aliquot (2 ml) of each protein sample was into separate wells of a 96 well plate.

2.

Add Bradford reagent (100 ml) to each sample. Ensure that no bubbles are introduced.

3.

Gently shake at room temperature for 5 min.

4.

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.

5.

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.

6.

The protein sample concentration can be calculated against the standard curve.

Immunoprecipitation Cross Linking

UV cross-linking and immunoprecipitation (CLIP) are two techniques utilized in molecular biology to study protein interactions with RNA or identify RNA modifications. Cross-linking allows for the proteins and RNAs to be covalently bonded, while immunoprecipitation uses antibodies to pull down the protein of interest. This method can be used with different types of RNA, including mRNAs, lncRNAs, and circRNAs.

There are several variations of CLIP, including:

  • Crosslinking and Immunoprecipitation of RNA-Binding Proteins (CLIP-seq): This method is used to study the RNA binding sites of a protein by immunoprecipitating the protein and then sequencing the co-immunoprecipitated RNAs.
  • Crosslinking and Immunoprecipitation of Modified RNAs (cLIP-seq): This method is used to study RNA modifications, such as m6A, by immunoprecipitating the modified RNA and then sequencing the co-immunoprecipitated RNAs.
  • Crosslinking and Immunoprecipitation of Native RNAs (CLIP-NAT): This method is used to study the native RNA-protein interactions by immunoprecipitating the protein and then sequencing the co-immunoprecipitated RNAs.

CLIP is a powerful tool that can be used to study RNA-protein interactions and RNA modifications. This method has been used to identify new RNA binding proteins, to map the binding sites of RNA binding proteins, and to study RNA modifications.

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