MT646 S9.6 Tandem IP with RNase treatment before final elution
Date: 10/21/21
Reference
08/29/18 - Orf2(MT302) Purification followed by RNAse treatment
October 14, 2021 - MT646 and MT302 Tandem IP with anti-ORF1 and S9.6 beads
October 5, 2021 - Effect of RNase treatment on RNA/DNA hybrid and L1 RNP (MT646)
Cell lines: MT646 High (08/26/21)
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 2x 150mg MT646 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 2x 150mg anti-ORF1 IP with 15ul of anti-ORF1 beads per IP reaction
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 anti-ORF1 beads
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 per 40ul of 20mM HEPES, pH7.4, 1% Tritonx-100, 125mM NaCl (final 200mM NaCl): for 25ul elution, add 100ul buffer
Split the diluted elution into 5x 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 (4x 50mg reactions)
2) MT646_mIgG (50mg)
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
Combine the FT from 4 replicates of S9.6 IP (total 100ul), take 25ul for Western and save the rest
Wash beads with 3x 200ul IP buffer (200mM NaCl)
RNase treatment on beads
Set up 4 reactions with S9.6 beads from 50mg IP:
1) 10ul L1-500 buffer + 0.5ul 2mg/ml BSA (Control)
2) 10ul L1-100 buffer with 8mM MgCl2, 1mM DTT + 0.5ul 5U/ul RNase H
3) 10ul L1-500 buffer with 8mM MgCl2, 1mM DTT + 0.5ul 5U/ul RNase H
4) 10ul L1-500 buffer + 0.5ul 2mg/ml RNase A/T1
• L1-100: 20mM HEPES, pH7.4, 1% Tritonx-100, 100mM NaCl
• L1-500: 20mM HEPES, pH7.4, 1% Tritonx-100, 500mM NaCl
Incubate @ RT 15’ with mixing
Collect the supernatant after 15’ for Western (“Sup”); Save these samples for Western
Wash beads with 3x 200ul of L1-500
Elute beads with 10ul 2% SDS/ @ 70°C for 5’, save the elution for Western (”E”)
Collect the eluate (“E”)
Check Input, FT, Sup and E by Western against anti-ORF1 and anti-ORF2 antibodies (Load 50% for anti-ORF1, and anti-ORF2 Western; Save the other 50%)
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
3) FT_S9.6
4) FT_mIgG
5) Sup_S9.6_BSA
6) Sup_S9.6_RNase H-100
7) Sup_S9.6_RNase H-500
8) Sup_S9.6_RNase A/T1
9) E_S9.6_BSA
10) E_S9.6_RNase H-100
11) E_S9.6_RNase H-500
12) E_S9.6_RNase A/T1
13) E_mIgG
14) Marker (not shown)
15) SDS_anti-ORF1 (SDS wash of anti-ORF1 beads, for 50mg IP)
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-mouse 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
Strip the top half of the membrane and reprobe it with anti-ORF2 antibody
Primary Ab: Rabbit anti-ORF2 antibody (clone 5-5, 1.03mg/ml), 1:500, RT for 2hr
Secondary Ab: ECL anti-rabbit HRP 1:10,000, RT, 1hr
10 second exposure, high sensitivity, auto tone
