Conjugation of anti-ORF1 and anti-ORF2 Dynabeads
Making anti-ORF2 beads, 10ug antibody per mg of beads Date: Feb.10, 2020
Dynabeads M-270 Epoxy (Invitrogen Dynal, Cat. No. 143.02D)
Antibody: anti-ORF2 (abcam clone 9-7), received on 9/12/19; concentration 1.5mg/ml by BCA assay; stored in 1x PBS with 0.01% NaN3
Weigh out 90mg of beads, pre-equilibrated in 0.1M NaPO4, pH7.4
Antibody mix: 10ug of anti-ORF2 antibody per mg of Dynabeads; final volume of antibody mix will be 20ul per mg of beads; final concentration of antibody mix is 0.5mg/ml
600ul anti-ORF2 antibody (900ug of antibody)
600ul 3M AmSO4 (final concentration 1M)
600ul 0.1M NaPO4, pH7.4
Mix antibody with 0.1M NaPO4, pH7.4 first in a 5ml Eppendorf tube
Add 3M AmSO4 one drop at a time to the tube while mixing
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)
The filtration step can be skipped if there is no antibody precipitation when making the antibody mix
Add the antibody mix to beads
Take 2ul of Ab mix before and after coupling, check them on gel.
Leave the tube on a thermomixer at 37°C, O.N. (18 - 24hr)
Wash the beads according to the Rout lab protocol Beads are resuspended in the following buffer for long-term storage at -20°C 50% Glycerol/1xPBS/0.5mg/ml BSA
Make 100ul aliquots of beads when stored at -20°C
Making anti-ORF1 and anti-ORF2 beads, 15ug antibody per mg of beads Date: Feb.11, 2020
Our previous results showed that when using 15ug instead of 10ug of anti-ORF1 antibody per mg of beads for conjugation, the resulting beads work better for IP (gave higher Orf1p yield) Wiki entry “12/11/19 - Reuse anti-ORF1 for beads conjugation”
We will use 15ug of anti-ORF1 per mg of beads here and also test this conjugation condition for anti-ORF2 beads (small scale test, 10mg beads) Antibody mix: 15ug of antibody per mg of Dynabeads; final volume of antibody mix will be 20ul per mg of beads
Anti-ORF1 beads
Anti-ORF1 antibody from Abmart (4H1), received on 11/18/19. Desalt 1ml of anti-ORF1 into 0.1M 0.1M NaPO4, pH7.4. Check the concentration by Bradford: 1.23mg/ml; Take 975ul (1500ug) to make 100mg of beads
80mg of beads: 1200ug of antibody; 1600ul antibody mix; final concentration of antibody mix is 0.75mg/ml
975ul anti-ORF2 antibody (150ug of antibody) 533ul 3M AmSO4 (final concentration 1M) 92ul 0.1M NaPO4, pH7.4 Anti-ORF2 beads
Antibody: anti-ORF2 (abcam clone 9-7), received on 9/12/19; concentration 1.5mg/ml by BCA assay; stored in 1x PBS with 0.01% NaN3; No desalting step needed
10mg of beads: 150ug of antibody; 300ul antibody mix; final concentration of antibody mix is 0.75mg/ml
100ul anti-ORF2 antibody (150ug of antibody) 100ul 3M AmSO4 (final concentration 1M) 100ul 0.1M NaPO4, pH7.4
Mix antibody with 0.1M NaPO4, pH7.4 first in a 5ml Eppendorf tube
Add 3M AmSO4 one drop at a time to the tube while mixing
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)
Add the antibody mix to beads
Leave the tube on a thermomixer at 37°C, O.N. (18 - 24hr)
Wash the beads according to the Rout lab protocol Beads are resuspended in the following buffer for long-term storage at -20°C 50% Glycerol/1xPBS/0.5mg/ml BSA
Make 100ul aliquots of beads when stored at -20°C
Check antibody mix on gel
1) Marker
2) Anti-ORF2 antibody (original, from abcam)_1ul
3) Antibody pellet from new Ab mix with 1.5M AmSO4_1ul
4) Ab mix pre-coupling #1_2ul
5) Ab mix post-coupling #1_2ul
6) Ab mix pre-coupling #2_2ul
7) Ab mix post-coupling #2_2ul
8) Ab mix pre-coupling #3_2ul
9) Ab mix post-coupling #3_2ul
10) BSA_50ng
11) BSA_150ng
Testing anti-ORF1_anti-ORF2 Beads and RRP6 powder Date: 02/12/20
Cell lines: LD401 heavy_05/02/14 (check anti-ORF1 and anti-ORF2 beads) RRP-3xFLAG_02/10/20 (check RRP6 expression after Dox induction)
Scale: 50mg for LD401 and 100mg for RRP6-3xFLAG Extraction/wash buffer: 20mM HEPES, pH7.4, 500mM NaCl, 0.5% Triton X-100
Weigh out powder in 2x 100mg LD401 heavy and 2x100mg RRP6-3xFLAG (100mg of old and 100mg of new powder); add 400ul of extraction buffer (1:4 w/v) to each tube, vortex to mix
2 Amp, 5x 2 sec (~20J total output); Operate in the cold room
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Combine the clarified cell extract of LD401 and split it equally into 6 aliquots (50mg equivalent each) with 2.5ul of anti-FLAG beads or 5ul of beads anti-ORF1 and anti-ORF2 beads
RRP6-3x FLAG lysate, each was used for 100mg IP with 10ul of beads
Set up the following IPs
1) LD401_anti-FLAG_2.5ul of beads
2) LD401_anti-ORF2_old beads
3) LD401_anti-ORF2_new beads_02/11/20 (10ug antibody per mg of beads)
4) LD401_anti-ORF2_new beads_02/12/20 (15ug antibody per mg of beads)
5) LD401_anti-ORF1_old beads
6) LD401_anti-ORF1_new beads
7) RRP6_anti-FLAG_10ul of beads (old powder)
8) RRP6_anti-FALG_10ul of beads (new powder)
IP @ 4°C for 30’ After IP, wash 3x 1ml extraction buffer
Elute with 15ul of 1x LDS, 70°C for 5’ with shaking
Collect LDS elution
Add 50mM DTT (final conc.)
Heat samples @ 70°C for 10’
Run elution on 4-12% Bis-Tris gels
1) Marker
2) E_LD401_anti-FLAG
3) E_LD401_anti-ORF2_Old
4) E_LD401_anti-ORF2_New-10
5) E_LD401_anti-ORF2_New-15
6) E_LD401_anti-ORF1_Old
7) E_LD401_anti-ORF1_New
8) Marker
9) E_RRP6_anti-FLAG_Old
10) E_RRP6_anti-FALG_New
11) BSA_50ng
12) BSA_150ng
Conclusion: From the test pullout, anti-ORF2 beads coupled under both conditions (10ug antibody per mg of beads or 15ug antibody per mg of beads) perform similarly. We will use the standard condition (10ug antibody per mg of beads) to make anti-ORF2 beads.
Dox induction of RRP6-3x FLAG cells
Monday 01/27/20 1 vial of stock -> 2x T75 flasks
Wednesday 2x T75 -> 2x T175 flasks
Friday use one T175 flask to make 5x 1.8ml stocks and the other one to seed 2x 500cm2 plates
Monday 2x 500cm2 plates ->16 plates
Wednesday 16 plates become 70% confluent, induce with 5ng/ml Dox
Thursday havest, 5.5g of BB’s (could induce @ 90% confluent next time)