Anti-ORF1 and mIgG IP for MS
Date: 09/29/20
• RNAse free L1 extraction buffer. 1:250 RNasin in EB, 1:1000 in wash. • 100mg scale with 20ul anti-ORF1 beads (20x12=240ul total)
12 samples total, 4 replicates from each cell line
• N2102Ep (1-4)
• NTERA2 (5-8)
• PA-1 PC (9-12)
1. Prep extraction buffer with RNAse inhibitor (1:250) and protease inhibitors (1:100); Add 1:4 (w/v) of extraction buffer to powder
2. Sonicate samples for 5x2 sec at 2 Amp; repeat once (energy output 100mg samples: ~20J)
3. Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R); save 10ul of input
4. During spin, wash 20ul of beads per sample with extraction buffer that does NOT have RNasin added (to conserve RNasin). (Beads stored in 1X PBS, 0.5ng/uL BSA, 50% glycerol.)
To wash beads, add 1mL wash buffer to each tube first, then add 20uL of bead slurry to each buffer-filled tube. Wash 3x with 1mL
6. Set up IP with 20ul of anti-ORF1 beads (made on 09/03/20) per 100mg reaction
7. IP@ 4°C for 30’
8. Save 10ul of FT after IP
9. Wash beads 3x 1ml with RNAsin containing wash buffer
10. Switch beads to fresh tubes at 2nd wash step
11. Elute in 25ul of 2% SDS/40mM Tris, pH8; save 2.5ul aside (10% of 100mg IP)
Combine the 10% elution of 4 replicates from the same cell line (~ 40mg IP) for gel analysis
12. Collect elution and put the tubes in speed vac to dry the samples down
13. Put the dry pellet in -20 freezer. Continue with S-trap protocol
Repeat the protocol above with mIgG beads
Check elution on 4-12% Bis-Tris gel with Sypro stain
1) Unstained marker
2) N2102Ep_anti-ORF1
3) N2102Ep_mIgG
4) NTERA_anti-ORF1
5) NTERA_mIgG
6) PA-1 PC_anti-ORF1
7) PA-1 PC_mIgG
8) BSA_50ng
9) BSA_100ng
10) Prestained marker
11) Unstained marker
2 second exposure, auto tone
The very dark band between 75 and 50 Kd in PA-1 PC lane is vimentin.
All samples were prepared using S-trap and sent to NL for MS
MS sample preparation using S-Trap Micro Ultra-High Recovery protocol
Resuspension solvent: 94.9:5.0:0.1 water:MeOH:FA
Resuspension volume: 20 uL
Injection volume: 5 uL as indicated in the Raw file’s name
MS method: uPU-60mingradTOP20-highload_Luciano_IP_gradient (for DDA)
uPU-60mingradTOP20-Hload_DIA_300_1500_window_15 (for DIA)
Link to RAW files: https://rockefeller.box.com/s/xqkobayui7uqrfu6mip1v5by9tuydtmr