Conjugation of anti-ORF2 Dynabeads
Date: 02/03/22
Dynabeads M-270 Epoxy (Invitrogen Dynal, Cat. No. 143.02D)
RU beads: lot # 00816517 (exp date 2021-02-28)
Antibody mix:
10ug of anti-ORF2 antibody per mg of dynabeads; final volume of antibody mix will be 20ul per mg of beads; 300ul total volume
Tube 1: Anti-ORF2: abcam (clone 5-5, lot 20211208AF1) received on 2/2/22; 1 mg/ml stored in 1x PBS with no NaN3. Take 150ug (150ul) to make 15mg of beads.
150 ul anti-ORF2 antibody_5-5 (2/2/22, 1 mg/ml)
100 ul 3M AmSO4 (final concentration 1M)
50 ul 0.1M NaPO4, pH7.4
Tube 2: Anti-ORF2: abcam (clone 9-7) received on 9/12/19; stored in 1x PBS with 0.01% NaN3. Desalted into 0.1M NaPO4, pH7.4. Final concentration 1.2mg/ml. Take 150ug (125ul) to make 15mg of beads.
125 ul anti-ORF2 antibody_9-7 (09/12/19, 1.2 mg/ml)
100 ul 3M AmSO4 (final concentration 1M)
75 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)
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
Check antibody mix before and after conjugation
Run 4-12% Bis-Tris gel, loading 2ul of antibody mix before and after conjugation
1) Marker
2) Antiobody mix_9-7_before
3) Antiobody mix_9-7_after
4) Antiobody mix_5-5_before
5) Antiobody mix_5-5_after
MT647 IP testing anti-ORF2 beads
Date: 02/04/22
Cell line: pMT647: ORF2p only ORFeus ORF2p-3xFlag CMV promoter in pCEP4 Puro
Extraction: 20mM HEPES, pH7.4, 1% Tritonx-100, 500mM NaCl (with protease inhibitors)
Scale and powder to beads ratio: 50mg per IP reaction with 5ul of beads
anti-ORF2 beads (9-7) Old_011421
anti-ORF2 beads (9-7) New_020422
anti-ORF2 beads (5-5) Old_052621
anti-ORF2 beads (5-5) New_020422
anti-FLAG beads Old_060419
Weigh out 200mg of cell powder, extraction buffer to each tube (1:4 w/v), vortex to mix
Sonicate 5 x 2 sec at 2 Amp; repeat once
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Split supernatant evenly into 4 aliquots (50mg each)
Set up 5x 50mg IP reactions with 5ul beads
1) anti-ORF2 beads (9-7) Old
2) anti-ORF2 beads (9-7) New
3) anti-ORF2 beads (5-5) Old
4) anti-ORF2 beads (5-5) New
5) anti-FLAG beads Old
IP @ 4°C for 30’
After IP, wash 3x 1ml extraction buffer
Switch beads to fresh tubes at 2nd wash step
Native elution with ORF2 di-peptides from 21st Century
ORF2 di-peptide stocks (MT5 and MT9) • 2.5mM in 50mM HEPEs, pH 7.4, 500mM NaCl, 0.5% Triton X-100
Dilute ORF2 di-peptide to 1mM with extraction buffer
3x FLAG peptide stock @ 5mg/ml inn TBS (50mM Tris, pH7.4, 150mM NaCl)
Incubate beads with 5ul of diluted peptide @ RT for 15’
1) anti-ORF2 beads (9-7) Old_MT9
2) anti-ORF2 beads (9-7) New_MT9
3) anti-ORF2 beads (5-5) Old_MT5
4) anti-ORF2 beads (5-5) New_MT5
5) anti-FLAG beads Old_3xFLAG
LDS wash of the beads after native elution
Wash beads with 10ul of 1.1x LDS for 10 min at RT w/ mixing
Transfer the LDS wash to a fresh tube (“LDS”)
Add 1x LDS and 50mM DTT (final concentrations) to gel samples as needed
Heat samples @ 70°C for 10’
Run 4-12% Bis-Tris gel, analyze elution and LDS wash
1) Marker
2) E_anti-ORF2 beads (9-7) Old_MT9
3) E_anti-ORF2 beads (9-7) New_MT9
4) E_anti-ORF2 beads (5-5) Old_MT5
5) E_anti-ORF2 beads (5-5) New_MT5
6) E_anti-FLAG beads Old_3xFLAG
7) LDS_anti-ORF2 beads (9-7) Old_MT9
8) LDS _anti-ORF2 beads (9-7) New_MT9
9) LDS _anti-ORF2 beads (5-5) Old_MT5
10) LDS _anti-ORF2 beads (5-5) New_MT5
11) LDS _anti-FLAG beads Old_3xFLAG
12) BSA_50ng
13) BSA_150ng
