Aim: Remove high molecular weight contaminants in his anti-ORF2 monoclonal antibody prep.
Materials:
Superose 200 increase 10/300 GL column (GE): 10kDa → 600 kDa separation range buffer: 1 x PBS Loaded 500 uL Flow rate: 0.25 mL/min
Fig. 1: Elution of protein standards on Superose200 increase 10/300 GL (GE)
Numbers correspond to the following MWs:
1: Thyroglobulin 669 kDa
2: Ferritin 440 kDa
3: Aldolase 158 kDa
4: Conalbumin 75 kDa
5: Ovalbumin 44 kDa
6: Carbonic anhydrase 29 kDa
7: Ribonuclease A 13.7 kDa
Results
Fig.2 FPLC trace of anti-ORF2 monoclonal antibody separation
● Peak is ~13 mL
● Eluting at roughly the same size as Aldolase (158 kDa)
● Some tapering, could be due to column interactions or heterogeneity of PTM’s on antibody.
Fig 3: SDS-PAGE of SEC fractions
Lanes: 1. MW marker (precision plus all blue, Bio-Rad, from Hua)
2. BSA 10 uL
3. BSA 15 uL
4. anti-ORF2 pre SEC
5. Fraction 2
6. Fraction 9
7. Fraction 10
8. Fraction 11
9. Fraction 12
10. Fraction 13
11. Fraction 14
12. Fraction 15
13. Fraction 16
14. Fraction 17
15. Fraction 18
● Fractions 12 - 16 contain protein (peak fractions 13 and 14) = in agreement with FPLC trace.
● 3 bands? Usually there’s a heavy chain at 50 kDa and a light chain at 25 kDa.
02/19/20 Concentrate purified anti-ORF2 antibody before making beads
Fractions 13 and 14, put tubes in speed vac, reduce the vol. from ~450 to 150ul each and combine
Fractions 9, 12, 13 (~1500ul total), combine, reduce vol to 600ul total in speed vac; split into 2 tubes (2x 300ul); add 100ul of 4M AmSO4, pH7.4 to each tube; put the tube back into the speed vol, reduce the vol to 150ul/tube (final AmSO4 is ~2.7M); Spin @ top speed for 10’; transfer the supernatant to different tube, resuspend the pellet in total of 100ul of 0.1M NaPhosphate, pH7.4
Make anti-ORF2 beads Date: Feb. 19, 2020
Combine all fractions and determine the concentration by Bradford Assay
Purified anti-ORF2: concentration 0.74mg/ml, total vol. 400ul (296ug total, use it to make 30mg of beads; ~10ug per mg of beads)
Recovered anti-ORF2 from previous conjugation (02/12/20): concentration 0.64mg, total vol. 300ul (192ug total, use it to make 20mg of beads; ~10ug per mg of beads)
Conjugation condition: 10ug of antibody per mg of beads; 20ul of antibody mix per mg of beads, 37°C overnight with mixing (24h total)
Wash beads with standard protocol
Store the beads in 50% glycerol/ 1xPBS / 0.5mg/ml BSA @ -20°C
Test anti-ORF2 beads Date: Feb. 20, 2020
Cell line: LD401 heavy_05/02/14
Extraction/wash buffer: 20mM HEPES, pH7.4, 500mM NaCl, 0.5% Triton X-100
Weigh out powder in 150mg LD401 heavy, add 600ul of extraction buffer, 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)
Split the clarified lysate evenly into 3 aliquots (50mg each) and set up IP with 5ul of anti-ORF2 beads (from 3 different batches)
1) LD401_anti-ORF2_Old_091719
2) LD401_anti-ORF2_022020a
3) LD401_anti-ORF2_022020b
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 @ 75°C for 10’
Run elution on 4-12% Bis-Tris gels
1) Marker
2) E1
3) E2
4) E3
5) Marker
6) Ab mix_pre coupling_2ul_purified
7) Ab mix_post coupling_2ul_purified
8) Ab mix_pre coupling_2ul_recovered
9) Ab mix_post coupling_2ul_recovered
10) BSA_50ng
11) BSA_150ng
12) Marker
13) 1ul anti-ORF2 (9-7) purified by Nat before concentrating (Fract.13)
14) 1ul anti-ORF2 (9-7) purified by Nat after concentrating (all combined)
15) 1ul anti-ORF2 (9-7) recovered from AbMix
Beads made on Feb. 19 (both from purified antibody or recovered antibody) seem to perform better than older batch. Will repeat anti-ORF2 IP with anti-FLAG IP as a control, make sure the difference we see here is not due to pipeting error of anti-ORF2 beads from different batches.