co-immunoprecipitation and immunoblotting

Sample preparation

1. Bone tissues or cells were collected at the indicated time and rinsed with PBS for 3 times.

2. Add suitable volume of ice-cold RIPA buffer or IP lysis buffer to the washed bone tissues or cells in a microcentrifuge tube.

RIPA buffer: 20 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM EGTA, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM sodium orthovanadate. Immediately before use, add the protease inhibitor mixture (Roche).

IP lysis buffer: 20 mM Tris HCl (pH 8.0), 137 mM NaCl, 1% NP-40, 2 mM EDTA. Immediately before use, add the protease inhibitor mixture (Roche).

To prepare bone tissues for protein analyses, cut them into pieces with scissor before add the lysis buffer, which were then homogenized using a homogenizer.

Optional: The samples could be sonicated in multiple (5~8) short bursts. Keep the samples immersed in an ice bath during sonication.

3. Incubate the tubes in an ice bath on a shaker for 30 min.

4. Centrifuge for 15 min at 16,000 rpm at 4°C in a microcentrifuge. Gently remove the tubes from the centrifuge and place on ice, aspirate the supernatant, and place in a fresh tube kept on ice; discard the pellet.

The protein concentration was determined using the Pierce BCA Protein Assay Kit (Cat. #23227) according to the manufacturer’s protocol.

Co-immunoprecipitation

1. On ice, in a microcentrifuge tube, add 50~100 μg total cell lysate plus the recommended amount of antibody (Check the antibody datasheet for recommended antibody concentration) and mix. 5~10 μg of the cell lysates of each sample could be subdivided and used as input control. The Anti-Flag (M2, Cat. #F1804) antibody was used.

2. Incubate the sample with the antibody over night at 4°C under gentle rotation.

3. Add 20 µl of resuspended Protein A/G PLUS-Agarose (Santa Cruz, Cat. #sc-2003). Cap tubes and incubate at 4° C on a rotating device for 5~6 hours. 

4. Collect immunoprecipitates by centrifugation at 2,500 rpm for 5 minutes at 4° C. Carefully aspirate and discard supernatant. BD Insulin Syringes with the BD Ultra-Fine^TM needle (31G) could be used to remove the supernatant more clearly.

5. Wash pellet 6 times with 1.0 ml wash buffer, each time repeating the centrifugation step above.

Wash buffer: 10 mM Tris (pH 7.4), 1mM EDTA, 1 mM EGTA (pH 8.0), 150 mM NaCl, 1% Trition X-100, 0.2 mM sodium orthovanadate. Immediately before use, add protease inhibitor mixture (Roche).

6. After final wash, aspirate and discard supernatant and resuspend pellet in 10 µl of 2X electrophoresis sample buffer [NuPAGE LDS Sample Buffer (4X), Cat. #NP0007].

7. Heat the samples at 70℃ for 10 min.

Optional: After heating, samples may be centrifuged to pellet the agarose beads followed by SDS-PAGE analysis of the supernatant.

Immunoblotting

1. Load the heated samples into the wells of the Invitrogen NuPAGE Bis-Tris protein gels, along with molecular weight markers.

2. Run the gel in MOPS buffer for 1–2 h at 80 V.

10X MOPS buffer recipe:

1) Prepare 800 mL of dH₂O in a suitable container.

2) Add 41.86 g of MOPS free acid to the solution.

3) Add 4.1 g of Sodium Acetate to the solution.

4) Add 3.72 g of Na₂EDTA to the solution.

5) Adjust solution to pH 7.0 using NaOH.

6) Add dH₂O until the volume is 1 L.

3. Transfer the proteins from the gels to nitrocellulose membrane in wet condition for 1.5–2 h at 150 mA in a tank.

4. Cut the nitrocellulose membrane according to the molecular weight of the targets.

5. Blocking the nitrocellulose membrane in 5% BSA (in TBST) for 1 h at RT.

6. Incubate the membrane with appropriate dilutions of primary antibodies in blocking buffer overnight at 4°C.

7. Wash the membrane 3 times in TBST, 15 min each.

8. Incubate the membrane with the recommended dilution of conjugated secondary antibody in blocking buffer at RT for 1 h.

9. Wash the membrane 3 times in TBST, 15 min each.

10. Develop the signal using the Pierce™ ECL Western Blotting Substrate and take images.

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