MT646 and MT302 Tandem IP with anti-ORF1 and S9.6 beads
Date: 10/14/21
Cell lines: MT646 High (08/26/21) and MT302 Dox 24-hour induction (8/26/21)
Extraction: 20mM HEPES, pH7.4, 1% Tritonx-100, 500mM NaCl
Scale of Tandem IP: 50mg with 5ul anti-ORF1 Epoxy beads (1st IP) and 1ul of S9.6 or mouse IgG Epoxy beads (2nd IP)
Weigh out 150mg MT646 and MT302 powder, add 600ul of extraction buffer (with protease inhibitors)
Sonicate @ 2Amp, 4x 2sec
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Collect the supernatant
Set up 150mg anti-ORF1 IP with 15ul of anti-ORF1 beads
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, 125mM NaCl (+ 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)
Split the diluted elution into 2x 25ul aliquots
Add 1ul of S9.6 or mouse IgG beads (Epoxy beads) to each tube; set up the following IPs (50mg IP with 1ul of epoxy beads)
1) MT646_S9.6
2) MT646_mIgG
3) MT302_S9.6
4) MT302_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 (Load 50% for anti-ORF1, and anti-ORF2 Western; Save the other 50% for S9.6 Western)
Western against anti-ORF2 and anti-ORF1 antibodies
Run Input, FT and E (25mg each) on 4-12% Bis-Tris gel
1) Marker
2) Input_MT646
3) FT_S9.6_MT646
4) FT_mIgG_MT646
5) E_S9.6_MT646
6) E_mIgG_MT646
7) Marker (not shown)
8) Input_MT302
9) FT_S9.6_MT302
10) FT_mIgG_302
11) E_S9.6_MT302
12) E_mIgG_MT302
13) Marker
14) SDS_anti-ORF1_MT646
15) SDS_anti-ORF1_MT302
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-FLAG Western
Primary Ab: mouse anti-FLAG antibody (Sigma F3165, 4mg/ml), 1:2000, 4C, overnight
Secondary Ab: ECL anti-rmouse 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
Note: I did not wash S9.6 and mIgG beads before
Spot and crosslink the other 50% of Input, FT and Elution on Nylon membrane and probe with S9.6 antibody
700/702 (DNA/RNA hybrid) titer - RNase (0.5ul) - RNase (1ul) - RNase (2ul) - RNase (5ul) Blank Blank
SDS wash of anti-ORF1 beads MT646anti-FLAG (50mg) Blank MT302 anti-FLAG (50mg) Blank Blank Blank
Primary Ab: mouse S9.6 antibody, 1:1000 (1ug/ml), 4°C overnight (Freshly diluted) Secondary Ab: HRP linked anti-mouse IgG, 1:10,000, RT for 1hr
10 minutes exposure, super sensitivity, auto tone (light image of the membrane shown on the right hand side)
1. There is some kind of contamination in the grid MT646 #2 (as shown in the picture of the membrane.) The black dot in that spot is not real.
2. Nylon membrane was cut below the 3rd row (MT302). The top panel was probed with fresh S9.6 antibody and the bottom panel was probed with old S9.6 antibody.