Conjugation of anti-ORF1, anti-ORF2 and mIgG Dynabeads
Date: 05/25/21
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): recovered antibody from old antibody mix, 1.76mg/ml in 0.1M NaPhosphate
Antibody mix: 15ug of anti-ORF1 antibody per mg of dynabeads; final volume of antibody mix will be 20ul per mg of beads, 600ul for 30mg of beads
256ul anti-ORF1 antibody (1.76mg/ml)
200ul 3M AmSO4 (final concentration 1M)
144ul 0.1M NaPhosphate, pH7.4
Anti-ORF2: abcam (clone 5-5): 1.03mg/ml in 1xPBS, 0.1% NaN3
Antibody mix: 10ug of anti-ORF2 antibody per mg of dynabeads; final volume of antibody mix will be 20ul per mg of beads, 600ul for 30mg of beads
300ul anti-ORF2 antibody (1.03mg/ml)
200ul 3M AmSO4 (final concentration 1M)
100ul 0.1M NaPhosphate, pH7.4
mIgG: Sigma I5381 dissolved in 1xPBS, 2mg/ml in 1xPBS
Antibody mix: 10ug of mIgG per mg of dynabeads; final volume of antibody mix will be 20ul per mg of beads, 600ul for 30mg of beads
150ul mIgG (2mg/ml)
200ul 3M AmSO4 (final concentration 1M)
250ul 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
For QC of the new beads
See May 27, 2021 - Testing new anti-ORF1 and mIgG beads and May 27, 2021 - Testing new anti-ORF2 beads and ORF2 dipeptides