MT289 and N2102Ep Tandem IP using anti-ORF1, S9.6 Protein G and mIgG Epoxy control beads
Date: 04/01/21
Scale: 25mg with 5ul anti-ORF1 or anti-FLAG Epoxy beads (1st IP) and 2.5ul of S9.6 Protein G beads or 1ul of Epoxy mouse IgG control beads (2nd IP)
Cell lines: MT289 Light (02/05/14): L1 expressor without FLAG tag and N2102Ep (02/09/21): L1 endogenous expressor; MT302 Light (02/05/14; L1RP Orf2-PPX-3xFLAG)
Extraction buffer: 20mM HEPES, pH7.4, 1% Tritonx-100, 500mM NaCl
Weigh out 75mg of MT289, 75mg of N2102Ep and 125mg of MT302 powder, add 4x vol (1:4 w:v) of extraction buffer (with protease inhibitors and RNasin 1:250)
Sonicate @ 2Amp, 4x 2sec
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Collect the supernatant, set up the following IP
1) MT289: 50mg of anti-ORF1 IP (10ul of anti-ORF1 beads)
2) N2102Ep: 50mg of anti-ORF1 IP (10ul of anti-ORF1 beads)
3) MT302: 50mg of anti-FLAG IP (5ul of anti-FLAG beads)
4) MT302: 75mg of anti-ORF1 IP (15ul of anti-ORF1 beads)
IP @ 4°C for 30’
Wash 3x 1ml extraction buffer
Transfer beads to fresh tubes after 2nd wash
Native elution @ RT for 15’ with shaking (10ul peptide for 75mg of IP)
1) MT289: 50mg of anti-ORF1 IP (7ul of 1mM ORF1 di-peptide) 2) N2102Ep: 50mg of anti-ORF1 IP (7ul of 1mM ORF1 di-peptide) 3) MT302: 50mg of anti-FLAG IP (7ul of 1mg/ml 3xFLAG) 4) MT302: 75mg of anti-ORF1 IP (10ul of 1mM ORF1 di-peptide)
Collect eluate
Wash beads with extraction buffer (half volume of the peptide)
Collect the wash and combine it with eluate 1) MT289: 50mg of anti-ORF1 IP (7+3.5=10.5ul total) 2) N2102Ep: 50mg of anti-ORF1 IP (7+3.5=10.5ul total) 3) MT302: 50mg of anti-FLAG IP (7+3.5=10.5ul total) 4) MT302: 75mg of anti-ORF1 IP (10+5=15ul total)
Save 5 for Western (“Input”; Load 50% on gel; equivalent to 12.5mg)
Dilute peptide elution 1:5 with 20mM HEPES, pH7.4, 1% Tritonx-100 (+ protease inhibitors) and do the S9.6 IP (final 200mM NaCl)
Dilute 5ul of elution with 20ul (1: 4) of 20mM HEPES, pH7.4, 1% Tritonx-100, 125mM NaCl (final 200mM NaCl) to set up the 2nd IP
Add 2.5ul of S9.6 or mouse IgG beads (old Protein G beads) to each tube; set up the following IPs (25mg IP with 2.5ul of protein G beads)
1) MT289 (anti-ORF1)_mIgG
2) N2102Ep_mIgG
3) MT302 (anti-FLAG)_mIgG
4) MT302 (anti-ORF1)_S9.6
5) MT302 (anti-ORF1)_mIgG
IP @ RT for 15’
Save the flow-through (25ul) for Western (“FT”); Load 50% of FT on gel (12.5mg)
Wash beads with 3x 200ul IP buffer (200mM NaCl)
Elute beads with 10ul 2% SDS/ @ 70°C for 5’
Collect the eluate (“E”)
Check Input (50%), FT (50%) and E by Western against anti-ORF1 and anti-ORF2 antibodies
Western against anti-ORF2 and anti-ORF1 antibodies Reference 05/31/18 - Orf2 (LD401) Tandem PO with S9.6 beads
Run Input, FT and E (25mg each) on 4-12% Bis-Tris gel
1) Marker
2) Input_MT289 (ORF1 dipeptide elution)
3) FT_mIgG_MT289
4) E_mIgG_MT289
5) Input_N2102Ep (ORF1 dipeptide elution)
6) FT_mIgG_N2102Ep
7) E_mIgG_N2102Ep
8) Input_MT302 (3x FLAG elution)
9) FT_ MT302_anti-FLAG_mIgG
10) E_ MT302_anti-FLAG_mIgG
11) Input_MT302_anti-ORF1 (ORF1 dipeptide elution)
12) FT_ MT302_anti-ORF1_S9.6
13) FT_ MT302_anti-ORF1_mIgG
14) E_ MT302_anti-ORF1_S9.6
15) E_ MT302_anti-ORF1_mIgG
Wet transfer 70V for 1.5h
Blocking: TBST/5% milk, RT 2hr
Cut the membrane between 75 and 50KD, use the top half for anti-ORF2 Western and bottom half for anti-ORF1 Western
Upper panel: Anti-ORF2 Western
Primary Ab: Rabbit anti-ORF2 antibody (Clone 5-5, 1.03mg/ml), 1:1000, 4C, overnight
Secondary Ab: ECL anti-rabbit HRP 1:10,000, RT, 1hr
Lower panel: Anti-ORF1 Western (Abmart, 2mg/ml)
Primary Ab: Mouse anti-ORF1 antibody, 1:5,000, 4°C overnight
Secondary Ab: ECL anti-mouse HRP 1:10,000, RT 1hr
10 second exposure, high sensitivity, auto tone
60 second exposure, high sensitivity, auto tone
