Completed in “RNA style” using nuclease-free reagents
Extraction buffer: 20mM HEPES, pH7.4, 1% Triton X-100, 500mM NaCl + P.I., + RNase Inhibitor @ 1:250
Wash buffer: 20mM HEPES, pH7.4, 1% Triton X-100, 500mM NaCl + P.I., + RNase Inhibitor @ 1:1000
Scale: 1600mg (8 x 200mg)
- 100mg of powder yields ~500ng of total protein via native elution
- 1600mg – 100mg for gel analysis = 1500mg of powder => 7.5ug of protein
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PART 1 - IPs and Crosslinking
1. Weigh out 8 x 200mg of powder, add 800ul of extraction buffer per tube in safe-lock 2mL tubes.
2. Sonicate 5x2 sec @ 4 Amp and then repeat a second time (20 total sec, total J will be 25-30)
3. Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
4. Combine clarified lysate from each of the tubes and then split evenly into 8 tubes, save 10ul aside for WB
5. Use the rest to set up 8X 200mg anti-FLAG IPs (using 10ul anti-FLAG beads per 100mg; 20ul beads for 200mg IP; this is based on Hua's recent titration of beads with this cell line 07/10/19 - LD401_anti-FLAG and anti-ORF1 beads titration)
6. Incubate @ 4°C for 30’
7. Prepare 1mg/ml 3x Flag peptide working sol'n: dilute 5mg/ml stock 1:5 in extraction buffer (20mM HEPES, pH7.4, 500mM NaCl, 1% Tritonx-100 + PI + RNAsin)
8. After IP, wash beads with 2x 1ml extraction buffer Transfer beads to new 2ml Eppendorf tube after 2nd wash
9. Wash beads once more with 1ml extraction buffer
10. Add 50ul of 1mg/mL 3xFLAG to each tube containing anti-FLAG beads (50ul per 200mg IP)
11. Incubate @ RT for 15' with shaking.
12. Transfer the elution to a fresh tube. Save the beads in one tube and put them at -20. These will be eluted a second time with 50 ul 1.1X LDS, but this can be done at the end.
13. Split the 3x FLAG elution into two aliquots: Save 1/16 (25uL, 100mg) of the 3x FLAG elution, this will be the “Input” fraction
The other 15/16 (375uL, 1500mg goes to anti-ORF1 tandem IP using 75uL anti-ORF1 beads (5ul anti-ORF1 beads per 100mg IP) split across 6 tubes (+/- 60-65ul 3xFLAG elution and 12.5uL beads per tube)
14. Incubate 3x FLAG elution with anti-ORF1 beads @ RT for 15’ with mixing
15. Save the supernatant after IP; this will be the “Sup” or “FT”
16. Wash the beads in each tube with 1x 1ml of wash buffer
17. Elute in 10uL ORF1 di-peptide diluted with extraction buffer to ~1mM (with protease inhibitors [no EDTA]) (P194712, A28 (see spec sheet) in 50mM HEPES, pH7.4, 500mM NaCl and 0.01% Triton)
18. Incubate @ RT for 15’ with mixing
19. Pool the elution and put on ice
20. Repeat elution step with 5uL buffer and add to the pooled elutions from the previous step this will be “E” fraction (~120 ul total).
21. Add the cross-linker at 2.5mM [Final] to the Elution and gently agitate the tubes in a thermal mixer at 750 rpm, 23°C for 25-30 min to cross-link the affinity isolated complexes
22. After cross-linking, quench the reaction by adding 50mM (final) Tris-HCl pH 7.5
24. Add 50mM final iodoacetamide (IAA) for cysteine alkylation. Incubate for 30 min in the dark.
25. Elute aFLAG and aORF1p beads with 1.1x LDS and incubate at 70C for 5' to collect what remains on the beads after peptide elution.
26. Add 4x LDS, and DTT to all samples and incubate at 70C for 10'.
27. Load samples on a 3-8% Tris-Acetate gel. Since sample was 100+ ul it was split across two lanes. First 50ul per lane was loaded, then gel was run until the sample was out of the well, after which another 40ul per lane was loaded.
Gel loading
1) marker
2) aORF1p Input/aFLAG E (1/16; 100mg)
3) aFLAG beads LDS elution (1/16; 100mg)
4) aORF1 FT (1/15; 100mg)
5) aORF1 Elution (1/15; 100mg)
6) aORF1 beads LDS elution (1/15; 100mg)
7) [space]
8) Elution (1/2)
9) Elution (1/2) 
The red areas were cut into 1mm^3 cubes and moved to Luciano/Dennis for subsequent digestion and MS analysis.