Conjugation of anti-ORF2 Dynabeads
Dynabeads M-270 Epoxy (Invitrogen Dynal, Cat. No. 14302D; lot #01138253)
Anti-ORF2: Abcam custom anti-ORF2
DAY ONE
Desalt into 0.1M NaPhosphate, pH7.4 using Zeba 7k desalting column. Perform BCA assay using BgG standard to determine concentration after desalting.
Antibody concentration after desalting was 1.26 mg/ml (total volume 660ul). We will use 10ug of anti-ORF2 per mg of beads. We will have enough antibody to make ~80mg of beads.
Allow beads to equilibrate to RT before opening the bottle / beginning
Weigh out appropriate amount of beads into appropriately sized tube and equilibrate in 0.1M NaPO4 pH 7.4. For NaPO4 equilibration step, ratio of beads to buffer is 300mg/16ml. Adjust accordingly.
To equilibrate beads in buffer, add appropriate amount of 0.1M NaPO4. Vortex 30 seconds then place on nutator or end-over-end rotator for 10 minutes. During the equilibration step, prepare the antibody mix.
After 10 minutes of mixing, place tubes on magnet, aspirate buffer then add same volume of NaPO4, vortex 30 seconds and aspirate again. Aspirate buffer and add antibody mix.
For 100mg of beads, ideal reaction volume is 2ml (20ul per mg of beads).
Antibody mix: 10ug of anti-ORF1 antibody per mg of dynabeads; final volume of antibody mix will be 20ul per mg of beads
660 ul anti-ORF1 antibody 533 ul 3M AmSO4 (final concentration 1M) 407 ul 0.1M NaPO4, pH7.4
To make the antibody mix, attach the tube holder to the vortex and set vortex to medium speed. Add NaPO4 to tube, then antibody, then place tube in holder on vortex on continuous medium mix. Add AmSO4 ONE DROP AT A TIME to avoid precipitation of antibody.
Skipped filtration step because I did not observe precipitation
Filter the resulting antibody mix using Costar Spin X column (0.45um)
• Load <500ul on each column (500ul capacity; The column can be use repeatedly for the same antibody mix. Load the column multiple times if the volume is more than 500ul)
• Spin 12,000g, 1min (16,000g maximum speed)
This filtration step can be skipped if there is no antibody precipitation observed while making antibody mix
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 37C, O.N. (18 - 24hr)
DAY TWO
Take 2ul of antibody mix after coupling for gel analysis
Keep the rest of the antibody mix @ -20C
Before beginning, make fresh triethylamine by adding 168ul stock to 11.156ml ddH2O in 15ml tube.
Wash the beads 1x 1ml 100mM Glycine-HCl, pH 2.5, put on and take off as quickly as possible
Wash the beads 1x 1ml 10mM Tris, pH 8.8
Wash the beads 1x 1ml fresh 100mM Triethylamine, put on and take off as quickly as possible
Wash the beads 5x 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 -20C; For 300mg of beads you need 2ml storage buffer; we will use 533.33ul/80mg
Test IP anti-ORF2 beads:
LINE1 extraction/wash buffer: 20mM HEPES pH 7.4, 500mM NaCl, 1% Triton X-100 Cell Line: N2102EP Scale: 50mg
Weigh out 50mg x3 of N2102EP powder
Add 200ul of extraction buffer plus protease inhibitor to each tube (1:4 w/v), vortex to mix
Sonicate 5 x 2 sec at 4 Amp;
Spin @ 20k rcf, 4°C for 10’, wash beads 3x 1ml wash buffer during spin
Collect the supernatant
Set up IP reactions with 10ul of beads each
IP @ 4°C for 30’
After IP, wash 3x 1ml extraction buffer
Switch beads to fresh tubes at 2nd wash step
Elute the beads with 10ul of 1.1x LDS, 70 °C for 5’ with mixing
Collect the eluate
Add DTT to 50mM to all samples and heat @70 °C for 10’
Run 4-12% Bis-Tris gel to analyze the elution, Blue Silver Stain
Test IP anti-ORF2 beads (second test of the same beads):
LINE1 extraction/wash buffer: 20mM HEPES pH 7.4, 500mM NaCl, 1% Triton X-100 Cell Line: LD401 (light) Scale: 50mg
Weigh out 50mg x3 of LD401 (light) powder
Add 200ul of extraction buffer plus protease inhibitor to each tube (1:4 w/v), vortex to mix
Sonicate 5 x 2 sec at 4 Amp;
Spin @ 20k rcf, 4°C for 10’, wash beads 3x 1ml wash buffer during spin
Collect the supernatant
Set up IP reactions with 10ul of beads each
IP @ 4°C for 30’
After IP, wash 3x 1ml extraction buffer
Switch beads to fresh tubes at 2nd wash step
Elute the beads with 10ul of 1.1x LDS, 70 °C for 5’ with mixing
Collect the eluate
Add DTT to 50mM to all samples and heat @70 °C for 10’
Run 4-12% Bis-Tris gel to analyze the elution, Blue Silver Stain
N2102EP Bead Test
Gel Load Order
LD401 Bead Test
Gel Load Order
areas under the peaks
peak area LD401
1 24203.8
2 22764.8
3 22765.0
peak area N2102EP
1 4636.2
2 1468.6
3 2514.2