Immunoprecipitation (IP) - 2. IP

Input When I’ve just started learning IP in grad school, the concept of input was hard to understand. So, I wanted to talk about input in detail.

When you do IP, you must have input. There are two main reasons why we use input. 1. By comparing the input band size and its intensity to the IP band size and its intensity, you can figure out the yield of IP product.

2. Input is also used as a positive control so that you will be able to figure out whether IP was not performed properly, or detection was not performed properly when you can’t detect anything from IP. Example 1. If you could see a band from input after western blotting while you were not able to see any band from IP, that means IP was not performed because of technical issues during IP or no connections between your targets. Example 2. If you were not able to see both input band and IP band from western blot results, that means there was a technical issue in western blotting.

When I’ve done RIP or ChIP, I’ve used to take 1% volume of whole lysate as input. For co-IP, I’ve used 30-60 ug of protein as input. You can adjust the amount of input according to your IP results. Since it’s hard to quantify when the western blot/dot blot/slot blot band are too saturated, using proper amount of input is important.

IP Method A IP with antibodies: Target Protein-Antibody reaction

<Antibody reaction>

<IP>

<Washing>

1. Target (sample): (pre-cleared) lysate + 2-5 ug target antibody Negative control: (pre-cleared) lysate + 2-5 ug IgG antibody Note1. In most cases, when you want to check the protein-protein, protein-RNA, or protein-DNA, you will overexpress a targeted protein to get distinguishable results. Especially tagged proteins (c-Myc, GFP, etc.) so that you can save money as tag antibodies are cheap. Note2. The amount of lysate and antibody can be adjusted according to your results. Note3. The host of IgG antibody and target antibody must be same.

2. Rotate the tubes at 4°C overnight. Note1. Make sure the sample is agitated properly. Note2. The target protein and the antibody will bind during this step.

3. Add 50 ul protein A/G beads (Santa-Cruz, cat. Sc-2003).

4. Rotate at 4°C for 1-4 h. Note. The beads will bind to the antibody during this step.

5. Centrifuge at 1,200g for 2 min at 4°C.

6. Remove sup. Note. Do not remove sup aggressively. Leave a little amount of sup so that you don’t take agarose beads.

7. Add 1 ml lysis buffer and rotate for 5 min for washing to get target protein-antibody-beads complex.

8. Centrifuge at 1,200g for 2 min at 4°C.

9. Repeat step 6-8, four times.

10. Remove sup after last centrifugation. Note. Remove sup as much as possible in this step while not taking beads.

11. Process elution.

Method B

IP with antibody-agarose beads:

1. Add 50 ul protein A/G beads (Santa-Cruz, cat. Sc-2003) to an ep tube.

2. Add 2-5 ug target antibody.

3. Rotate at 4°C for 1-4 h.

4. Centrifuge at 1,200g for 2 min at 4°C.

5. Add 1 ml of lysis buffer and rotate for 5 min for washing.

6. Repeat step 4-5, twice.

7. Add (pre-cleared) lysate.

8. Rotate at 4°C for 1-4 h.

9. Centrifuge at 1,200g for 2 min at 4°C.

10. Remove sup.

11. Add 1 ml lysis buffer and rotate for 5 min.

12. Centrifuge at 1,200g for 2 min at 4°C.

13. Repeat step 10-12, four times.

14. Process elution.