1. T cells are grown as usual (plate-bound until 72 h, expanded in IL-2 until day 6)
2. Restimulation: Plate ~70 million cells in 2 10” Greiner cell culture plates. Leave ~30 million cells unstimulated in flask or 10” plates.
After 16h
4. Add 10um MG-132 to restimulated and unstim cells (1:1000 from 10mM MG-132 stock). Wait 4 hours
3. Harvest cells (restimulated cells may need to be scraped off) into falcons, spin down
4. Wash with 1ml cold PBS and transfer into eppendorf, spin down. Remove almost all PBS and mix up the pellet with the remaining PBS.
5. To pellet add 100ul preboiled lysis buffer (25 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 1% SDS, 0.1% NP-40, 1 mM phenylmethylsulfonyl fluoride, 25 μg/ml of aprotinin, 25 μg/ml of leupeptin, 10 mM NaF, 8 mM β-glycerophosphate, 1 mM sodium vanadate, 1 mM dithithreitol, 10μM MG-132, and 5 mM NEM; NEM is a powder that lives in the fridge and a very small mass is needed, so I usually make more of this buffer than necessary)
6. Vortex and incubate at 95° for 5mins
7. Add 900 ul NP-40 buffer (25 mM HEPES pH 7.5, 150 mM NaCl, 0.2% NP-40, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 25 μg/ml of aprotinin, 25 μg/ml of leupeptin, 10 mM NaF, 8 mM β-glycerophosphate, 1 mM sodium vanadate, 1 mM dithithreitol, 10μM MG-132, and 5 mM NEM) to dilute stringent buffer 1:10.
8. The lysate will be really thick and goopy now. As quickly as possible, pass lysate through 18g needle and syringe, then through 23g needle. Hopefully the lysate should be less viscous at this point. Centrifuge at 4° 14000rpm for 10 minutes.
9. Harvest supernatant into cold eppendorf tube on ice.
Meanwhile: prepare Protein G magnetic beads
Per sample, you’ll need 30ul beads (10ul for preclear, 20ul for IP)
1. Take volume of beads into eppendorf and onto magnet.
2. Suck off liquid, and wash the beads 3x with 0.1% BSA, putting the tube back on the magnet and sucking off liquid in between.
3. Resuspend beads in same volume as they were in to begin with in 1% BSA.
4. Leave tube on RT nutator for 30 mins.
5. Back on magnet and wash 3x with 0.1% BSA, again putting the tube back on the magnet and sucking off liquid in between.
6. Resuspend beads in same volume as they were in to begin with in 0.1% BSA.
Back to IP:
10. Add 10ul beads to each sample. The rest of the beads can sit in the fridge until tomorrow.
11. Leave tube on nutator in cold room (ideally the one in the Chapman lab’s cold room) for 1 h.
12. Tube back on magnet and harvest supernatant. This is your lysate. If you’re doing a negative control IgG IP, this is the step to split your lysate in half. This is also the time to take 4% of your recovered volume for input. Add 5x sample buffer with β-Me like normal protein sample. One half of lysate gets 10ul Wako α-mouse Ago2 antibody, and one half gets 10ul α-mouse IgG.
14. Leave tube on nutator in cold room (ideally the one in the Chapman lab’s cold room) overnight.
The next day…
15. Take beads from fridge (they settle so you’ll need to shake them up). Add 20ul beads to each sample. Return to cold nutator for 1 hr.
16. Put beads onto magnet and suck off liquid.
17. Wash beads 3x with RIPA (50mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 1% Triton X 100 or NP-40)
18. Resuspend each sample in 1x sample buffer (5x sample buffer with β-Me into RIPA to 1x)
19. Boil samples at 95° for 5 min.
20. Put tubes back on magnet and harvest liquid. This is your IP sample.
Western Blot time!
-You’ll need to run 2 different gels for the 4% input and the IP. I use the 4-15% Biorad Tris-HCl gradient gels. I like Biorad #1611158 (though I think they’re being replaced with some other version). These have 10 wells that hold almost 50ul, which is useful because your 4% input volume will be quite high.
-Gels can be run as normal.
Transfer!
-Ubiquitin blots seem to look better if they’re transferred using wet transfer. We use the Biorad Mini Trans-blot system and follow the instructions for setting up the transfer. The transfer buffer is different than the usual one we use. Instead make 10x Tris-glycine transfer buffer
30.3g Tris
144.1 g Glycine
up to 1 L with water---store at 4°
-To make 1x:
85mL 10x tris-glycine buffer
150mL methanol
up to 1L with water--- store at 4°, can be reused 3-5x.
-Perform wet transfer at 400mA for 1.5h. Make sure to keep it cold, so use an ice pack inside the apparatus and do the whole thing on ice.
-The 4%input gel could probably also be done with wet transfer, but Ago2 blots don’t seem to look as nice on PVDF, so I do this one with semi-dry.
Antibodies:
Anti-mouse ubiquitin (P4D1) from Covance 1:500
-this signal will be super strong and basically will prevent you from seeing any other antibodies after. If you want to look at ubiquitin on your input blot, make sure to do actin and/or Ago2 before ubiquitin
anti-mouse Ago2 from Cell signaling 1:1000
anti-mouse Actin from Sigma 1:10000