Conjugation of anti-ORF1 Dynabeads for N2102EP Drug Assay
Dynabeads M-270 Epoxy (Invitrogen Dynal, Cat. No. 143.02D); Lot #00886548
We need 1280ul beads for the drug assay (64 samples x 20ul/samples); we have ~400ul of antiORF1 beads from Hua; We will make 100mg of beads using 15ug antibody/mg of beads = 1500ug antibody needed
Buffer exchange antibody using 7k MWCO Zeba column into 0.1M NaPhosphate pH 7.4
Check the concentration by BCA before continuing
r2 =0.993
2 replicates: 1.054mg/ml and 911.1ug/ml
Antibody mix: 15ug of anti-ORF1 antibody per mg of dynabeads; final volume of antibody mix will be 20ul per mg of beads, 2ml for 1000mg of beads
1.5ml anti-ORF1 antibody
500ul 4M AmSO4 (final concentration 1M)
0 0.1M NaPhosphate, 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
Wash the beads 1x 1ml 100mM Glycine-HCl, pH 2.5; on and off as fast as possible
Wash the beads 1x 1ml 10mM Tris, pH 8.8
Wash the beads 1x 1ml fresh 100mM Triethylamine; on and off as fast as possible
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
Test IP to confirm successful bead conjugation
N2102EP Abmix Input, Abmix FT, Huas beads, old ERIBA beads, new ERIBA beads
All batches of beads tested were pooled and re-aliquoted prior to use in the IP.