General Protocols
Protocol for crosslinking IgG to Dynabeads Protein A or G
- Solutions
- BS3 Conjugation Buffer
- BS3 Quenching Buffer: 1M Tris HCl (pH 7.5)
- Protocol
1. Prepare 100 mM BS3 in Conjugation Buffer (Stock Solution).
Proceed to making a 5 mM solution by diluting in Conjugation Buffer, 250 µL is required per sample.
- Note: BS3 Stock and Conjugation solutions must be freshly made prior to use!
2.Wash the Ig-coupled Dynabeads Protein A or Protein G twice in 200 µL Conjugation Buffer. Place on magnet and discard supernatant.
3.Resuspend the Dynabeads in 250 µL 5 mM BS3.
4.Incubate at room temperature for 30 min with tilting/rotation.
5.Quench the cross-linking reaction by adding 12.5 µL Quenching Buffer
6.Incubate at room temperature for 15 min with tilting/rotation.
7.Wash the cross-linked Dynabeads three times with 200 µL PBST (or IP buffer of your choice). Place on magnet and discard supernatant.
8.Proceed with your IP and antigen elution (starting from step 2.3 of the Immunoprecipitation protocol).
Immunoprecipitation
We have been using Dynabeads Protein G
Required buffers:
Wash Buffer: PBS with 0.02% Tween
Elution Buffer: 200mM Glycine pH 2.5
1. Prepare a tube with your diluted antibody
a.Dilute 1-10ug of your antibody in 200ul PBS-T.
b.How much you add depends on how strong your antibody is
2. Vortex the Dynabeads vial until re-suspended.
3. Pipette 50 ul of beads into an Eppendorf tube.
4. Place the tube in the magnetic stand to separate the beads from the solution.
5. Remove and discard the supernatant.
6. Add your antibody dilution to the tube with the beads.
7. Incubate at room temperature, with rotation, for 10 minutes.
- During this time the antibody should bind to the Dynabeads.
8. Place the tube in the magnetic stand and remove the supernatant.
9. Remove from the magnetic stand and add 200 ul PBS-T to suspend the beads.
10 Break up the pellet by gently pipetting. This will wash the beads.
11. Place the tube back on the magnetic stand and remove and discard the supernatant.
12. In a separate tube, prepare your protein sample.
- Aim for 250-500ug of protein, with a volume of 100-1000ul. (Nanodrop or do a BCA beforehand so you know how much you are adding)
- It is okay to dilute your protein in PBS-T to get it to an appropriate volume.
13. Add your protein dilution to the tube with the beads and gently pipette to resuspend.
14. Incubate with rotation at room temperature for 10 minutes.
- During this time the antigen will bind to the antibody that is stuck to the beads.
- The amount of time you incubate depends on the affinity of the antibody
- The longer you incubate for, the more non-specific binding you risk
15. Remove from the roller and place on the magnetic stand.
16. Remove the supernatant and keep in a separate tube.
- This is the flow through
- You can run it out on a gel to make sure your protein was okay (if you don’t have anything show up on a silverstain for the flowthrough, you probably didn’t have enough protein and probably won’t have anything show up on the eluent)
17. Take the tube with the beads off of the magnet. Add 200 ul PBS-T and gently pipette to wash.
18. Put back on magnetic stand, and remove and discard the supernatant.
19. Repeat the wash step a minimum of 3 times (recommend about 6 washes)
20. After your final wash, re-suspend the beads in 100ul PBS-T and transfer to a clean tube.
*This is to make sure you do not have any proteins that may have gotten stuck to the side of the tube (and not the beads) in your final elution.
21. Eluting the sample
22. If you are going to run your sample out on a gel:
- Place the tube on the magnetic rack and remove the supernatant.
- Add 30 ul of laemelli buffer.
- Add 5 ul of reducing agent.
- Boil at 100° for 10 minutes.
- Spin the tube down to get rid of condensation
- Put the tube on the magnetic rack and gently mix the supernatant by pipetting
- Pipette sample directly onto gel
If you are going to do an in-solution digestion:
- Place the tube on the magnetic rack and remove the supernatant.
- Add 60 ul of 200 mmol Glycine pH 2.5.
- Incubate, for 2 min, with rotation, at room temperature.
- Place the tube on the magnetic stand and transfer supernatant to a separate tube.
- At this point the pH of the glycine should have caused the antigen-ab complex to come off the beads.
- If you want to take an aliquot of this sample to run on a gel, make sure to add Laemelli and a reducing agent (and boil)
