N2102Ep Tandem IP using anti-ORF1 and anti-DNA-RNA hybrid Protein G beads (S9.6)
Date: 03/22/21
Scale: 50mg with 10ul ORF1 (1st IP) and 10ul of S9.6 Protein G beads or 10ul of mouse IgG Protein G control beads
Cell line: N2102Ep
Extraction buffer: 20mM HEPES, pH7.4, 1% Tritonx-100, 500mM NaCl
Weigh out 150mg N2102Ep (2/9/21) powder, add 600ul of extraction buffer (with protease inhibitors and RNasin 1:250)
Sonicate 5 sec at setting 3
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Collect the supernatant, set up 150mg anti-ORF1 IP with 30ul 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 20ul of 1mM ORF1 di-peptide @ RT for 15’ with shaking
Collect eluate
Wash beads with 10ul of extraction buffer
Collect the wash and combine it with eluate (30ul total)
Save 10ul for Western (anti-ORF1 elution, “Input”)
02/12/18 - Testing S9.6 antibody against RNA/DNA hybrid
05/31/18 - Orf2 (LD401) Tandem PO with S9.6 beads
Split the rest into 2x 10ul aliquots
Dilute the ORF1 dipetide elution 1:5 with 20mM HEPES, pH7.4, 1% Tritonx-100 (+ protease inhibitors) and do the S9.6 IP (final 100mM NaCl)
Reference 05/31/18 - Orf2 (LD401) Tandem PO with S9.6 beads
10ul elution + 40ul of 20mM HEPES, pH7.4, 1% Tritonx-100
Add 10ul of S9.6 or mouse IgG beads (old Protein G beads) to each tube
IP @ RT for 15’
Save the flow-through (50ul) for Western (“FT”)
Wash beads with 3x 1ml IP buffer (100mM NaCl)
Elute beads with 10ul 2% SDS/ @ 70°C for 5’
Collect the eluate (“E”)
Check Input, FT and E by Western against anti-ORF2 and anti-ORF1 antibodies
Western against anti-ORF2 and anti-ORF1 antibodies
Reference 05/31/18 - Orf2 (LD401) Tandem PO with S9.6 beads
Run 4-12% Bis-Tris gel
1) Marker
2) Input_5ul (50% of 50mg)
3) FT_100_S9.6 (50% of 50mg)
4) FT_100_mIgG (50% of 50mg)
5) Marker (not shown)
6) E_100_S9.6 (50% of 50mg)
7) E_100_mIgG (50% of 50mg)
8) Space
9) E_MT302_anti-ORF1 (10mg)
10) Marker (not shown)
Wet transfer 70V for 1.5h
Blocking: TBST/5% milk, RT 2hr
Cut the membrane between 75 and 50KD, use bottom half for anti-ORF1 Western
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
High sensitivity, 10 second exposure, auto tone
Note: when we do anti-FLAG, anti-ORF1 tandem followed by anti-ORF1 and anti-ORF2 Western, we did not see any ORF1p or ORF2p signal in elution from mIgG control. But ORF1p was detected in elution from control beads.
Western against S9.6 antibody Reference 02/12/18 - Testing S9.6 antibody against RNA/DNA hybrid
Cut Hybond N+ nylon membrane (GE) to appropriate size. Mark and draw grid using pencil on the membrane when it is dry. (The membrane we have is from GE Healthcare, Amersham Hybond-N, Cat # RPN 203B)
Spot the samples before soaking in TBST
Spot various amount of elution (10% and 40%) onto membrane, and let it sit on a piece of filter paper, air dry for 5 min.
Soak the membrane in TBST for 1 min. Lay it flat on clean paper towel for 5-10 min until it is damp but not wet.
Crosslink in the UV Stratalinker 2400 (Stratagen) at the “Auto Crosslink” setting (1200 uJoulesX100). If the membrane is dry, soak and dry again as in step 7 before crosslinking. (Crosslinking is most efficient when the membrane is damp but not wet.)
Block the membrane in 5% milk /TBST for one hour at room temperature.
Incubate membrane with 1:500 S9.6 to detect DNA-RNA hybrids.
Primary Ab: mouse S9.6 antibody (Kerafast, ENH001, 1.25mg/ml),1:1000, 4°C overnight Secondary Ab: HRP linked anti-mouse IgG, 1:10,000, RT for 1hr
Light image of the blot
Ultra sensitivity, 2 min exposure, auto tone
Ultra sensitivity, 20 min exposure, without auto tone, overlay with the blot
Note: I believe the signal from Input (anti-ORF1 native elution) is real although it took long time to develop, and I tried ultra-sensitivity. The spots where the signal showed up were exactly in the middle of the grid where I loaded the samples. Signal from 4ul is much stronger than signal from 1ul sample. Not sure if SDS elution of S9.6 and mIgG beads will work here.