Anti-ORF2 IP (MT646, MT647, LD564)
Date: 02/02/22
Cell lines:
pMT646: ORFeus ORF2p-3xFlag (clean tags) CMV promoter in pCEP4 Puro (S221)
pMT647: ORF2p only ORFeus ORF2p-3xFlag (clean tags) CMV promoter in pCEP4 Puro
pLD564: L1RP ORF2p-3xFlag
Extraction Buffer: 20mM HEPES, pH7.4, 500mM NaCl, 1% Triton X-100 (v/v)
Scale: 5ul of anti-FLAG, anti-ORF2 (5-5 and 9-7) beads per 50mg IP
Weigh out 150mg of each kind of powder; add 600ul of extraction buffer
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) MT646_anti-FLAG
2) MT646_anti-ORF2 (5-5)
3) MT646_anti-ORF2 (9-7)
4) MT647_anti-FLAG
5) MT647_anti-ORF2 (5-5)
6) MT647_anti-ORF2 (9-7)
7) MT647_anti–ORF1 (negative control)
8) LD564_anti-FLAG
9) LD564_anti-ORF2 (5-5)
10) LD564_anti-ORF2 (9-7)
11) LD401_anti-ORF2 (9-7)
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) MT646_anti-FLAG
2) MT646_anti-ORF2 (5-5)
3) MT646_anti-ORF2 (9-7)
4) MT647_anti-FLAG
5) MT647_anti-ORF2 (5-5)
6) MT647_anti-ORF2 (9-7)
7) MT647_anti–ORF1 (negative control for ORF2p on Coomassie gel)
8) LD564_anti-FLAG
9) LD564_anti-ORF2 (5-5)
10) LD564_anti-ORF2 (9-7)
11) LD401_anti-ORF2 (9-7)
12) Marker
13) BGG_100ng
14) Anti-ORF2 (5-5)_200ng
15) Anti-ORF2 (5-5)_400ng
Western
Wet transfer, 70V for 2h
Block the membranes in TBST/5% milk @ RT, 2hr
Cut the membrane in half, between 75 and 50KD
Upper panel: Rabbit anti-ORF2 Western (Clone 11)
• Primary Ab: Rabbit anti-ORF2 antibody (Clone 11, 0.445mg/ml), 1:500, 4°C, O.N.
• Secondary Ab: ECL anti-rabbit HRP 1:5,000, RT, 1hr
Lower panel: Anti-ORF1 Western (Abmart, 4H1, 2mg/ml)
• Primary Ab: Mouse anti-ORF1 antibody, 1:5,000, 4°C, O.N.
• Secondary Ab: ECL anti-mouse HRP 1:10,000, RT 1hr
1) Marker
2) MT646_anti-FLAG
3) MT646_anti-ORF2 (5-5)
4) MT646_anti-ORF2 (9-7)
5) MT647_anti-FLAG
6) MT647_anti-ORF2 (5-5)
7) MT647_anti-ORF2 (9-7)
8) MT647_anti-ORF1
9) LD564_anti-FLAG
10) LD564_anti-ORF2 (5-5)
11) LD564_anti-ORF2 (9-7)
12) LD401_anti-ORF2 (9-7)
13) Marker (not shown)
30 seconds exposure, high sensitivity, auto tone
