Conjugation of anti-ORF1 Dynabeads
Date: 04/08/21
Dynabeads M-270 Epoxy (Invitrogen Dynal, Cat. No. 143.02D)
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. 1.4ml). Will make 110mg 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
1400ul anti-ORF1 antibody (desalted, 1.22mg/ml)
733ul 3M AmSO4 (final concentration 1M)
67ul 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
Testing new anti-ORF1 beads
Date: 04/09/21
Cell line: MT289 Light (05/02/14; to test new anti-ORF1 beads_04/09/21)
Extraction Buffer: 20mM HEPES, pH7.4, 500mM NaCl, 1% Triton X-100 (v/v)
Scale: 30mg for MT289 anti-ORF1 IP (6ul of anti-ORF1 beads per 30mg IP)
Weigh out 60mg of MT289 powder; add 540ul of extraction buffer (1:9 to increase the reaction volume of 30mg IP)
Vortex to mix; put the tubes on ice after resuspending the powder
Sonicate 2 Amp, 4x 2 sec
After sonication, spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Collect the clarified lysate
Set up the following IP reactions:
1) MT289_anti-ORF1 (30mg; 500mM NaCl buffer)_Old beads_04/06/21
2) MT289_anti-ORF1 (30mg; 500mM NaCl buffer)_New beads_04/09/21
IP @ 4°C for 30’ with rotation (cold room)
Wash beads with 3x 1ml extraction buffer
Transfer the beads to fresh tubes during 2nd wash
After the 3rd wash, spin down briefly and remove any remaining liquid
Elute in 10ul of 1.1x LDS for 10 min at RT w/ mixing
Place on a magnet; transfer the supernatant to a fresh tube
Add 50mM DTT (final conc.)
Heat samples @ 70°C for 10’
Run 4-12% Bis-Tris gel
1) Marker
2) Anti-ORF1 Abmix before coupling_2ul
3) Anti-ORF1 Abmix after coupling_2ul
4) E_MT289_Old anti-ORF1 beads
5) E_MT289_New anti-ORF1 beads
6) BSA_50ng
7) BSA_150ng
