Immunoprecipitation Protocol for Rab5a Antibody (NB120-13253)

Immunoprecipitation Protocol

Immunoprecipitation (IP) is a common useful immunochemical technique. When coupled with SDS-PAGE, IP can reveal a number of important characteristics of an antigen, including quantity, molecular weight, rate of synthesis or degradation, or interactions with other molecules. In IP, antigen is extracted in lysis buffer and antibodies are incubated with the lysate to form immune complexes. These complexes are isolated using a solid phase Protein A or Protein G matrix which facilitates separation and washing, yielding purified immune complexes that can be analyzed by immunoblotting.

Required Materials:

Protein A/G

Required Buffers:

1. 2X reducing sample buffer
2. 5% BSA/PBS (w/v)
3. RIPA buffer (or any lysis buffer): 50 mM Tris (pH 8.0), 150 mM NaCI, 0.1% SDS, 1.0% Tergitol, 0.5% sodium deoxycholate. Complete with protease inhibitor cocktail tablets
4. Reducing sample buffer such as Laemmli Buffer.

Basic 2X Laemmli Buffer contains:

4% SDS
20% glycerol
10% 2-mercaptoethanol
0.004% bromphenol blue
0.125 M Tris HCl
The solution has a pH of approximately 6.8.

Required Equipment:



Rehydrate Protein A/G Agarose/Sepharose:
1. Mass out ~100 mg of Protein A/G into a microfuge tube (enough for 10 reactions).
2. Rehydrate the 100 mg of Protein A/G with ~ 1 mL PBS.
3. Mix and incubate at 4C for 1 hour.
4. Wash the Protein A/G three times in 1 mL PBS, microcentrifuging at appropriate speed for ~10 seconds and aspirating the supernatant in between washes. (If Protein G is used, start with step 4)

Block the Protein A/G with 5% BSA/PBS:
5. Resuspend the Protein A/G with an equal volume of 5%BSA/ PBS to make a 50% Protein A/G slurry.
6. Incubate the Protein A/G at 4C on a rocker for 2 hours or overnight. (Blocking prevents binding of non-specific proteins, which form covalent bonds with Protein A/G beads.)

Pre-clear Cell Lysate:
7. Prepare complete RIPA buffer by adding the protease inhibitor tablet into RIPA buffer.
8. Thaw the appropriate amount of lysate; dilute the lysate to 2 mg/mL with the complete RIPA buffer.
9. Add 50 uL of 50% Protein A/G to the lysate
10. Rotate the mixture at 4C for 1 hour.
11. Micro-centrifuge the pre-cleared lysate at appropriate speed for 20 seconds to pellet the Protein A/G.
12. Carefully transfer the pre-cleared lysate to a clean tube and then transfer ~ 20 uL of the pre-cleared lysate to a labeled tube as the lysate positive control.

Forming and purifying the immune complex:
13. Add 200 uL of the pre-cleared lysate (plus any desired controls) to 200 uL of Complete RIPA buffer.
14. 2.5 ug of test antibody to tube #2 and #3. Note that the optimal amount will vary by antibody.
15. Rotate the reaction mixture of antigen and antibody at 4C overnight.
16. Next day, add 60 uL of Protein A/G to each tube and rotate at 4C for 2 hours.
17. Collect IP complex by micro-centrifuging the mixture for 30 seconds at appropriate speed, and aspirate off supernatant.
18. Wash all reactions 5 times with 1 mL of Complete RIPA buffer. To wash, resuspend the Protein A/G with the buffer, vortex briefly, centrifuge at appropriate speed for 30 seconds, and aspirate the supernatant (make sure to aspirate all the supernatant at the last wash).

19. Resuspend the Protein A/G with 50 uL of 2X reducing sample buffer. Prepare the lysate positive control by mixing 20 uL of the pre-cleared lysate with 5 uL of 5X reducing sample buffer.
20. Boil samples for 5 minutes. Micro-centrifuge briefly to pellet the Protein A/G.
21. Load ~15 uL of the supernatants, pre-cleared lysate, nonprecleared lysate on SDS gels. Samples can be stored at -20C if the gel will be run later.
22. Transfer the gels for Western blotting analysis.
23. For Western blotting, refer to Western blotting Protocol on page 252.

Troubleshooting for nonspecific background problems:

Add saturating amounts of competitor proteins. BSA, gelatin, acetone powders, or 5% nonfat dry milk are commonly used. Store Protein A/G in 5% BSA.
Spin the lysate at 100,000g for 30 minutes prior to the addition of the antibody to remove aggregated proteins.
Try a different antibody.
Centrifuge the antibody at 100,000g for 30 minutes and titrate.
Use some stringent washes. Try 1.0 M NaCI, 0.5 M lithium chloride, 1 M potassium thiocyanate, 0.2% SDS, or 1% Tween-20. Also alternating wash buffers from high salt to low salt or different detergents may help. Try washing with distilled water for one wash.
Increase the number of washes. Leave the solid phase in the wash buffers for 10 minutes at each wash.
Make certain the lysates are not frozen before use.
If specific proteins remain, remember that your antigen may consist of more than one polypeptide chain.