3. Immunoprecipitation
Immunoprecipitation can be performed using antibodies by different methods. The use of these methods is based on the requirements of end users. The first approach (4.2 Method A) is to mix antibody with protein sample and allowing it to interact with protein followed by addition of Protein A/G support. This method yields high purity of protein however the antibodies are also co-eluted with protein of interest which sometime creates difficulties in western blot detection. The second approach (4.2 Method B) is to bind antibody to the Protein A/G beads and then mix with the antigen. This method gives lesser yield than first one and avoids the problem of co-elution of antibodies. The third approach is to mix antibody, antigen and beads together; this method gives lowest yield and purity.
1) Method A
Immunoprecipitation with antibodies in solution
a. On ice, in a micro centrifuge tube add 10-50 μg cell lysate plus the recommended amount of antibody (see below). These amounts will be chosen depending on the abundance of the protein and the affinity of the antibody for the protein. Typically in a pilot experiment a fixed amount of protein is precipitated by increasing amounts of antibody.
Check the antibody datasheet for recommended antibody concentration. As a guideline use:
1-5 μl polyclonal antiserum
1μg affinity purified polyclonal antibody
0.2-1 μl ascites fluid (monoclonal antibody)
20-100 μl culture supernatant (monoclonal antibody)
b. Incubate the sample with the antibody between 1-12 hr (overnight) at 4°C, preferably under gentle agitation or rotation. The length of the incubation period depends on the amount of protein and affinity properties of the antibody.
c. Meanwhile prepare the Sepharose beads. If using a monoclonal antibody choose protein G-coupled Sepharose beads. If using a polyclonal antibody, protein A-coupled Sepharose beads are usually suitable (please refer to ‘Choosing the protein beads’ table below).
If the beads come as a powder, incubate 100 mg of beads in 1 ml 0.1 M PBS, wash for 1 hr so they swell up, then centrifuge, remove the supernatant and discard. Add 1 ml PBS 0.1% BSA, mix for 1 hr using eppendorf rotator and rinse in PBS 2 X. Remove the supernatant and add 400 μl of buffer made with protease inhibitors (can be the same as the lysis buffer). The slurry is now ready for use. It can be stored at 4°C for a few days; for longer periods keep the beads in PBS with 0.02% azide (rinse the beads extensively on the day of use and make up in fresh lysis buffer). You can also buy pre-swollen beads as slurry ready for use.
It is advisable to use pipette tips with the end cut off to prevent damage to the beads. IgM antibody: Do not use protein-A or protein-G conjugated beads. Use anti IgM coupled protein A or Protein G beads. The IgM will then bind to the beads by binding to the anti-IgM antibody. |
Click here to view the procedure using IgM antibodies:
d. Mix the slurry well and add 70-100 μl of the beads to each sample. Always keep samples on ice. Beads will tend to stick to the sides of the tip so try to minimize the movement in the pipette and use a tip cut 5 mm from the top.
e. Incubate the lysate beads mixture at 4°C under rotary agitation for 4 hr (the optimal incubation time can be determined in a preliminary experiment).
f. Follow the steps in section 5 wash.
2) Method B
Immunoprecipitation with Antibody-Agarose conjugate
a. To prepare Protein A or G agarose/Sepharose beads, follow the step 3 in method A.
b. To the microcentrifuge tubes add approximately 70-100 µl of slurry of Protein A-, or G-, or L-agarose conjugate.
c. Add 10 µl of primary antibody. Use the dilution recommended on the antibody datasheet for IP as a guideline.
d. Incubate the antibody-beads mixture for 1-4 hours at 4°C by gently mixing the mixture on a suitable shaker.
e. Centrifuge at 1,000-3,000 g for 2 minutes at 4°C and discard the supernatant.
f. Add 1 ml lysis buffer to the mixture by keeping gentle agitation and then centrifuge at 3,000 g for 2 minutes at 4°C. Repeat this washing step twice.
g. After washing the beads and antibody mixture, add 10-50 μg of cell lysates.
h. Incubate the lysate-beads/antibody conjugate mixture at 4°C under rotary agitation between 4 hours to overnight as required (The optimal incubation time can be determined in a preliminary experiment).
i. At the end of the incubation, continue with wash steps given in section 5.
4. Wash
1) When the lysate-beads/antibody incubation time is over centrifuge the tubes, remove the supernatant from the beads and discard. The complex of interest should now be specifically bound to the antibody coating the beads.
2) Wash the beads with washing buffer or lysis buffer 3 times, to remove non-specific binding. For each wash, mix the beads gently with wash buffer, centrifuge at 4°C and remove the supernatant which can be discarded.
3) Finally, ensure to carefully remove as much wash buffer as possible from the beads. The complex is now ready for elution from beads.
Using loading buffer is the harshest elution method, and will also elute any non-covalently bound antibodies and antibody fragments, which will appear on western blot gels. Antigens can be gently eluted with a glycine gradient (up to 1 M) to reduce the amount of eluted antibody. |
Click here to view separate procedure for cross-linking antibody to Sepharose.
5. Elution
One of the three common methods can be used to elute the complex from the beads. The SDS buffer is the harshest buffer which will also elute non-covalently bound antibodies and antibody fragments along with the protein of interest on the other hand Glycine buffer gently elutes the protein with reduced amount of eluted antibody.
1) Glycine Buffer Elution:
In this procedure the complex can be eluted from the beads by acidification. A glycine buffer containing 0.1-0.2 M Glycine pH 2.0-3.0 can be used. The low pH of glycine buffer helps to weaken the interaction between the antibody and the beads. This method is advantageous as beads can be reused after removal of the glycine buffer. However the eluted sample should be immediately neutralized with Tris, pH 8.0-8.5. After standard IP, follow these steps for glycine elution:
a. Elute the beads (50 µl) 3 X with 150 µl 0.2 M glycine pH 2.6 (1:1) by incubating the sample for 10 minutes with frequent agitation before gentle centrifugation.
b. Pool the eluate and neutralize by adding equal volume of Tris pH 8.0.
c. Repeat these steps 1-2 times and collect all the eluate.
d. Neutralize the beads by washing 2 X with 150 µl lysis buffer (without detergent) and pool with eluate.
e. Run the samples on a western blot to check the precipitation of proteins.
2) SDS Buffer Elution
In SDS elution the Ag-Ab complex is eluted from the beads by heating or boiling samples in loading buffer with denaturant SDS. This method is advantageous because the extraction method is highly efficient and the resulting sample is more concentrated.
a. Elute 50 µl of beads by heating in 50 µl of 2 X SDS without DTT for 10 min at 50°C.
b. Pellet beads, transfer supplement to a new tube and add DTT at 100 mM (Elution 1).
c. Add 50 µl 2 X SDS Buffer with DTT to pelleted beads (Elution 2).
d. Boil the elution samples for 5 min and analyze content of the sample by western blot.
Generally, there should be target protein in both elutions 1 and 2 collected above although amounts in each will be variable; Elution 2 will have more IgG contamination than Elution 1.
3) Urea Buffer Elution:
In this method the complex is eluted from the beads by the chaotrope urea. Beads should be resuspended in 2-5 volumes of urea elution buffer (6 – 8 M Urea, 20 mM Tris pH 7.5, and 100 mM NaCl). The purified complexes have now been released into the supernatant which should be collected from above the beads. This method is advantageous for mass spectrometry because the sample can be digested by proteolytic enzymes.
a. Wash beads with pre-urea wash buffer (50 mM Tris pH 8.5, 1 mM EGTA, 75 mM KCl). Remove all residual supernatant.
b. Add 100 µl urea elution buffer and rotate for 30 min at room temperature with frequent agitation before gentle centrifugation.
c. Repeat this process at least twice more to ensure that the entire captured complex has been released from the beads. Pellet beads and remove urea to a new tube.
d. Add 3 X sample buffer to run on a gel in western blot.
References:
1. Harlow, Ed, and David Lane. Using Antibodies. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press, 1999.
2. Bonifacino, Juan S. et al. Current Protocols in Immunology 8.3.1-8.3.28, New York: John Wiley, 2001.
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