Test anti-ORF2 beads Date: Feb. 26, 2020
Cell line: LD401 light_05/02/14
Extraction/wash buffer: 20mM HEPES, pH7.4, 500mM NaCl, 0.5% Triton X-100
Weigh out powder in 2x 100mg LD401 heavy, add 400ul of extraction buffer 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)
Split the clarified lysate evenly into 3 aliquots (50mg each) and set up IP with 2.5ul of anti-FLAG or 5ul of anti-ORF2 beads (from 3 different batches)
1) LD401_anti-FLAG
2) LD401_anti-ORF2_Old_021120
3) LD401_anti-ORF2_022020a
4) 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
Add 50mM DTT (final conc.) to each sample
Heat samples @ 70°C for 10’
Run elution on 4-12% Bis-Tris gels
1) Marker
2) E_anti-FLAG
3) E_anti-ORF2_old
4) E_anti-ORF2_new_purified Ab
5) E_anti-ORF2_new_recovered Ab
6) BSA_50ng
7) BSA_150ng
