Conjugation of anti-ORF1 and Dynabeads
Date: 09/22/20
Dynabeads M-270 Epoxy (Invitrogen Dynal, Cat. No. 143.02D)
RU beads: lot # 00816517 (exp date 2021-02-28)
Check the concentration by Bradford Assay and use desalted antibody to make beads
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 (total vol. 1.22ml). Will make 50mg 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, 1000ul for 50mg of beads
410 ul anti-ORF1 antibody (desalted, 1.22mg/ml); 333 ul 3M AmSO4 (final concentration 1M); 257 ul 0.1M NaPO4, pH7.4
Also, recovered anti-ORF1 from old antibody mix, 400ul in 0.1M NaPhosphate, pH7.4 @ 1.29mg/ml. Will make another 50mg of beads use recovered antibody.
388 ul anti-ORF1 antibody (recovered, 1.29 mg/ml); 333 ul 3M AmSO4 (final concentration 1M); 279ul 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
Anti-ORF1 IP with N2102Ep testing anti-ORF1 beads
Date: 09/24/20
Cell line: N2102Ep_011320
Extraction: 20mM HEPES, pH7.4, 1% Tritonx-100, 500mM NaCl (with protease inhibitors)
Scale and powder to beads ratio: 50mg per IP reaction with 10ul of anti-ORF1(4H1)
Anti-ORF1 beads: Control old anti-ORF1 beads_021220 (Old-1: without sonication; Old-2: with sonication) New anti-ORF1_092320 (New-1: uncoupled beads from RU, New-2: anti-ORF1 recovered from old Ab)
Weigh out 50mg and 150mg of cell powder, extraction buffer to each tube (1:4 w/v), vortex to mix
50mg powder: vortex to mix, no sonication
150mg powder: Sonicate 5 x 2 sec at 2 Amp; repeat once
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Split supernatant from 150mg cell extract evenly into 3 aliquots
Set up 4x 50mg IP reactions with 10ul anti-ORF1
1) N2102Ep_anti-ORF1_Old-1 (no sonication) 2) N2102Ep_anti-ORF1_Old-2 3) N2102Ep_anti_ORF1_New-1 4) N2102Ep_anti-ORF1_New-2
IP @ 4°C for 30’
After IP, wash 3x 1ml extraction buffer
Switch beads to fresh tubes at 2nd wash step
Elute with 10ul LDS @ RT for 10’ with mixing
Elute again with 10ul LDS @70 °C for 5’ with mixing
Add DTT to 50mM to LDS elution and heat @70 °C for 10’
Run 4-12% Bis-Tris gel to analyze the elution
1) N2102Ep_anti-ORF1_Old-1_RT
2) N2102Ep_anti-ORF1_Old-1_70C
3) N2102Ep_anti-ORF1_Old-2_RT
4) N2102Ep_anti-ORF1_Old-2_70C
5) N2102Ep_anti-ORF1_New-1_RT
6) N2102Ep_anti-ORF1_New-1_70C
7) N2102Ep_anti-ORF1_New-2_RT
8) N2102Ep_anti-ORF1_New-2_70C
9) Marker
10) BSA_50ng
11) BSA_150ng
12) Ab mix_anti_ORF1_RU_before_fresh antibody
13) Ab mix_anti_ORF1_RU_after_fresh antibody
14) Ab mix_anti_ORF1_RU_before_recovered antibody
15) Ab mix_anti_ORF1_RU_after_recovered antibody
