MT289 and N2102Ep Tandem IP using anti-ORF1 and anti-DNA-RNA hybrid Protein G beads (S9.6)
Reference 06/05/18 - Orf2 (MT302) Tandem IP using anti-FLAG and S9.6 beads
Date: 03/30/21
Scale: 25mg with 5ul anti-ORF1 Epoxy beads (1st IP) and 2.5ul of S9.6 Protein G beads or 2.5ul of mouse IgG Protein G control beads (2nd IP)
Cell lines: MT289 (02/05/14): L1 expressor without FLAG tag and N2102Ep (02/09/21): L1 endogenous expressor
Extraction buffer: 20mM HEPES, pH7.4, 1% Tritonx-100, 500mM NaCl
Weigh out 75mg MT289 and 75mg of N2102Ep powder, add 300ul 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 75mg anti-ORF1 IP of MT289 and N2103Ep with 15ul of anti-ORF1 beads (10ul anti-ORF1 beads per 50mg IP)
IP @ 4°C for 30’
Wash 3x 1ml extraction buffer
Transfer beads to fresh tubes after 2nd wash
Elute with 10ul of 1mM ORF1 di-peptide @ RT for 15’ with shaking
Collect eluate (10ul each)
Wash beads with 5ul of extraction buffer
Collect the wash and combine it with eluate (15ul total)
Save 5ul for Western (anti-ORF1 elution, “Input”)
Dilute the ORF1 dipetide elution 1:5 with 20mM HEPES, pH7.4, 1% Tritonx-100 (+ protease inhibitors) and do the S9.6 IP (final 200mM NaCl)
10ul elution + 40ul of 20mM HEPES, pH7.4, 1% Tritonx-100, 125mM NaCl (final 200mM NaCl)
For each cell line, split the diluted elution into 2x 25ul aliquots
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_S9.6
2) MT289_mIgG
3) N2102Ep_S9.6
4) N2102Ep_mIgG
IP @ RT for 15’
Save the flow-through (25ul) for Western (“FT”); Put the FT in speed vac to reduce the volume from 25ul to ~10ul
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, FT 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
3) FT_S9.6_MT289
4) FT_mIgG_MT289
5) Input_N2102Ep
6) FT_S9.6_N2102Ep
7) FT_mIgG_N2102Ep
8) Marker (not shown)
9) E_S9.6_MT289
10) E_mIgG_MT289
11) E_S9.6_N2102Ep
12) E_mIgG_N2102Ep
13) Space
14) E_MT302_anti-ORF1 (10mg)
15) Marker (not shown)
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 seconds exposure, standard sensitivity
Long exposure to show ORF2p
60 seconds exposure, high sensitivity, auto tone
10 minutes exposure, super sensitivity, auto tone
