Immunoprecipitationand dephosphorylation
Rong Zhao, Danish Uddin, Yi-Ting Chen, Sept 2016
Antibodybinding and antigen immunoprecipitation, by two methods:
• Plan A is adapted from the Borchelt lab protocol and starts by incubating the sample with the primary antibody before adding the beads. This method takes one day longer than Plan B due to the overnight sample+antibody incubation.
• Plan B is based on the manufacturer's recommended protocol included with the Novex Dyanbeads kit and binds the antibody to the beads before adding the sample. This method can be completed in half a day.
In our initial tests, Plan A was slightly more efficient than Plan B, and so remains the preferred method for the lab.
Plan A
Day 1:
1. Make 10 ml of RIPAbuffer:
PBS containing:
5 mM EDTA
0.5% Igepal
0.5% deoxycholate
0.2% SDS
1x protease inhibitor(Roche complete ULTRA tablets EASYpack #05892970001, one tablet/10 ml)
1x phosphatase inhibitor (Roche PhosSTOP #04906845001, one tablet/10ml)
2. Vortex and fully thawtissuehomogenates to room temperature. Bring 100 g of brain homogenate to 500 l with IP buffer. The approximate protein concentration of a 10% tissue homogenate made in SDS-containing buffer (ie, RIPA buffer or high-SDS RIPA buffer) is 10 mg/ml.
3. Boil the diluted homogenate solution for 5 min (you can use a 105º C heat block for this), followed by centrifugation at 15,000 g for 5 min at RT to remove insoluble material. Cool briefly, then transfer to a cleanEppendorf tube.
4. Add 2 g of primary antibodyto the homogenate. Incubate the sample + antibody mixture overnight at 4º C with gentle agitation such as on a Nutator. Wrap the lid with Parafilm to prevent leakage.
Day 2:
5. Carefullyresuspend the Dynabeads (ThermoFisher #10006D) by gently shaking the vial by hand.Transfer 50 l (1.5 mg) of bead slurry to a clean Eppendorf tube.
6. Place tubes into the DynaMag to separate the beads from the solution. Carefully pipet off the supernatant and discard.
7. Remove the tube from the magnet and add 200 l of provided Antibody binding and washing buffer (ABW buffer). Resuspend the beads by gently shaking by hand. Spin briefly to remove solution from the lid if needed.
8. Place tubesinto the magnet and discard supernatant.
9. Add the sample + antibody solution to the beads and gently resuspend by hand.
10. Incubate the beads and sample + antibody solution for 1 hr at RTwith rotation (Nutator).
11. Place tubesinto the magnet and remove supernatant. Save this solution to a clean tube if sample is limited - it can be reused for other antigens.
12. Wash the antigen-bound beads 3 times, using 200 l ABW buffer foreach wash. Mix gently by hand for each wash.
13. Resuspend the bound beads in 100 l ABW buffer and transferthe beadsuspension to a clean tubeto avoid eluting proteins bound to the tube wall.
14. Store the tube on ice while awaiting completion of other samples.
Plan B
Steps 1-3 are the same as above.
4. Carefully resuspend the Dynabeads (ThermoFisher #10006D) by gently shaking the vial by hand. Transfer 50 l (1.5 mg) of bead slurry to a clean Eppendorf tube.
5. Place tubes into the DynaMag to separate the beads from the solution. Carefully pipet off the supernatant and discard.
6. Dilute 2 g primary antibody in 200 l ABW buffer and pipet this into the tube containing the beads. Resuspend the beads in the diluted antibody solution by gentle mixing.
7. Allow antibody to bind protein A beads by incubating for 1 hr at RT with rotation (Nutator).
8. Place the tube into the magnetand discard the supernatant.
9. Remove the tube from the magnet and add 200 l of ABW buffer. Resuspend the beads by gently shaking by hand. Spin briefly to remove solution from the lid if needed.
10. Place tubesinto the magnet and discard supernatant.
11. Add the diluted tissue homogenatetothe beads and gently resuspend by hand.
12. Incubate 1 hr at RT with rotation to allow antigen binding.
13. Place tube into the magnetand remove the supernatant. Save this solution to a clean tube if sample is limited - it can be reused for other antigens.
14. Wash the antigen-bound beads 3 times, using 200 l ABW buffer for each wash. Mix gently by hand for each wash.
15. Resuspend the bound beads in 100 l ABW buffer and transfer the bead suspension to a clean tubeto avoid eluting proteins bound to the tube wall.
16. Store the tube on ice while awaiting completion of other samples.
Phosphatase treatment
1. Following immunoprecipitation, place the tubesinto the DynaMag and discard the supernatant.
2. Bound antigens are dephosphorylated in situby adding240 units (0.6l) of Lambda protein phosphatase (NEB # P0753S) in 120 l 1X NEB buffer and 1X MnCl2(both provided by NEB with the enzyme) for 1 hr at 30°C. Mix the beads gently by hand several times during the incubation to ensure the enzyme has access to the bound proteins.
3. Place tubesinto the magnet and discard thesupernatant.
4. Wash the antigen-bound beads 3 times, using 200 l ABW buffer for each wash. Mix gently by hand for each wash.
5. Resuspend the bound beads in 100 l ABW buffer and transfer the bead suspension to a clean tubeto avoid eluting proteins bound to the tube wall.
Antigen elution (Denaturing elution)
NOTE: Immunoprecipitated proteins can be stored frozen before running a gel. To freeze samples after IP, add the provided elution buffer and your own Laemmli or Tricine sample buffer (complete with ME for denaturing gels) to the washed beads and freeze the slurry at -20º C for up to a few days or -80º C for longer periods.
1. Place tubesintothe magnet and discardsupernatant.
2. Add 15l of the provided Elution buffer and 20 l of your own 2X Laemmli or Tricine sample buffer containing -mercaptoethanol.
3. Resuspend the beads by gentle hand mixing. Avoid foaming.
4. Incubate for 10 min at 70º C to dissociate the antigen-antibody complex.Use a heat block set to ~75º C for this step.
5. Centrifuge briefly, then place the tubes into the magnet and transfer the supernatant to a clean tube.
Western blot for APP-CTF (or other peptides <20 kDa)
1. Load the eluted sample onto a 16.5% Tris-tricine gel.
2. Electrophorese at 75 V for 20 min then 100 V for 5 hr until smallest MW marker is ~1" from the bottom of the gel.
3. Transfer the gel to nitrocellulose (BioRad Transblot Turbo transfer pack #1704159) for 15 min using the BioRad TransBlot Turbo.