Conjugation of anti-ORF1 and anti-ORF2 Dynabeads Date: 04/05/21
Dynabeads M-270 Epoxy (Invitrogen Dynal, Cat. No. 143.02D)
Desalt anti-ORF1 (4H1) into 0.1M NaPhosphate, pH7.4; S9.6 antibody can be used for conjugation directly
Check the concentration by Bradford Assay before conjugation
Anti-ORF1: Abmart (5310-1-4/4H1) received on 11/18/19; desalt into 0.1mM NaPhosphate, pH7.4 using Zeba 7k desalting column. Antibody concentration after desalting was 1.2mg/ml. Will make 60mg of beads use freshly desalted antibody.
Antibody mix: 15ug of anti-ORF1 antibody per mg of dynabeads; final volume of antibody mix will be 20ul per mg of beads, 900ug of antibody, 1200ul antibody mix for 60mg of beads
750 ul anti-ORF1 antibody (desalted, 1.2mg/ml)
400 ul 3M AmSO4 (final concentration 1M)
50 ul 0.1M NaPO4, pH7.4
S9.6: anti-DNA-RNA Hybrid [S9.6] antibody from Kerafast, cat # EN0001 (PBS, 0.05% (w/v) Sodium Azide
Antibody mix: 10ug of S9.6 antibody per mg of dynabeads; final volume of antibody mix will be 20ul per mg of beads, 100ug of antibody enough to make 10mg of beads; 200ul of antibody mix (put the thermomixer in 37C incubator
90 ul anti-ORF1 antibody (1.2 mg/ml, 108ug; 1vial of 100ug antibody)
66 ul 3M AmSO4 (final concentration 1M)
44 ul 0.1M NaPO4, pH7.4
Take 2ul of antibody mix before coupling for gel analysis
Add antibody mix to beads pre-equilibrated with 0.1M NaPO4, pH7.4; mix well
Leave the tube on a thermomixer at 37°C, O.N. (18 - 24hr); Put the thermomixer in 37°C incubator
Take 2ul of antibody mix after coupling for gel analysis
Keep the rest of the antibody mix @ 4°C
Wash the beads 1x 1ml 100mM Glycine-HCl, pH 2.5
Wash the beads 1x 1ml 10mM Tris, pH 8.8
Wash the beads 1x 1ml fresh 100mM Triethylamine
Wash the beads 4x 1ml 1x PBS (5’ each)
Wash the beads 1x 1ml 1x PBS + 0.5% Triton X-100, 5’
Wash the beads 1x 1ml 1x PBS + 0.5% Triton X-100, 15’
Resuspend beads in 50% Glycerol/1xPBS/0.5mg/ml BSA for long-term storage at -20°C
Testing anti-ORF1 and S9.6 beads
Date: 04/06/21
• MT289 anti-ORF1 IP testing anti-ORF1 (37.5mg with 7.5ul of beads, Aquastain)
• MT289 and N2102Ep Tandem IP testing S9.6 beads
Cell lines: MT289 Light 05/02/2014 and N2102Ep (09/09/21)
Extraction: 20mM HEPES, pH7.4, 1% Tritonx-100, 500mM NaCl (with protease inhibitors)
Scale of Tandem IP: 25mg with 5ul anti-ORF1 Epoxy beads (1st IP) and 1ul of S9.6 or mouse IgG Epoxy 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 2x 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 the following:
1. Anti-ORF1 IP to test new anti-ORF1 beads (37.5mg with 7.5ul of anti-ORF1 beads)
• MT289_anti-ORF1 (new, 04/06/21)
• MT289_anti-ORF1 (old, 01/08/21)
IP @ 4°C for 30’
Wash 3x 1ml extraction buffer
Transfer beads to fresh tubes after 2nd wash
Elute with 10ul of 1.1x LDS @ RT for 10’ with shaking
Add 50mM (final concentration) DTT to LDS elution and heat @70 °C for 10’
Run 4-12% Bis-Tris gel to analyze antibody mix before and after conjugation and MT289 anti-ORF1 elution
1) Maker
2) Ab mix_anti_ORF1_before
3) Ab mix_anti-ORF1_after
4) Ab mix_S9.6_before
5) Ab mix_S9.6_after
6) BSA_50ng
7) BSA_150ng
8) E_MT289_anti-ORF1_Old (accidentally load some BSA in this lane)
9) E_MT289_anti-ORF1_New
2. Tandem IP testing S9.6 beads using mIgG beads as control
Set up 75mg anti-ORF1 IP of MT289 and N2103Ep with 15ul of anti-ORF1 beads
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 1ul of S9.6 or mouse IgG beads to each tube; set up the following IPs (25mg IP with 1ul of epoxy 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
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) E_S9.6_MT289
6) E_mIgG_MT289
7) Marker (not shown)
8) Input_N2102Ep
9) FT_S9.6_N2102Ep
10) FT_mIgG_N2102Ep
11) E_S9.6_N2102Ep
12) E_mIgG_N2102Ep
13) 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 second exposure, high sensitivity, auto tone
60 second exposure, high sensitivity, auto tone
