Testing new anti-ORF1 beads
Date: 05/27/21
Cell line: MT289 Light (05/02/14)
Extraction Buffer: 20mM HEPES, pH7.4, 500mM NaCl, 1% Triton X-100 (v/v)
Scale: 25mg for MT289 anti-ORF1 IP (5ul of anti-ORF1 beads per 25mg IP)
Weigh out 100mg of MT289 powder; add 900ul of extraction buffer (1:9 to increase the reaction volume of 25mg IP)
Vortex to mix; put the tubes on ice after resuspending the powder
Sonicate 2 Amp, 4x 2 sec
After sonication, spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Collect the clarified lysate
Set up the following 4x 25mg IP reactions:
1) anti-ORF1_091120
2) anti-ORF1_052621
3) mIgG_002219
4) mIgG_052621
IP @ 4°C for 30’ with rotation (cold room)
Wash beads with 3x 1ml extraction buffer
Transfer the beads to fresh tubes during 2nd wash
After the 3rd wash, spin down briefly and remove any remaining liquid
Elute in 10ul of 1.1x LDS for 10 min at RT w/ mixing
Place on a magnet; transfer the supernatant to a fresh tube
Add 50mM DTT (final conc.)
Heat samples @ 70°C for 10’
Run 4-12% Bis-Tris gel
1) Marker
2) Anti-ORF1 Abmix before coupling_2ul
3) Anti-ORF1 Abmix after coupling_2ul
4) Anti-ORF2 (5-5) Abmix before coupling_2ul
5) Anti-ORF2 (5-5) Abmix after coupling_2ul
6) Marker
7) mIgG Abmix before coupling_2ul
8) mIgG Abmix after coupling_2ul
9) E_MT289_Old anti-ORF1 beads
10) E_MT289_New anti-ORF1 beads
11) E_MT289_Old mIgG beads
12) E_MT289_New mIgG beads
13) BSA_50ng
14) BSA_150ng
