N2102Ep and NTERA anti-ORF2 IP with rIgG controls
Date: 10/20/20
• RNAse free L1 extraction buffer. 1:250 RNasin in EB, 1:1000 in wash.
• Cell lines: N2102Ep and NTERA; use old MT302 anti-ORF1 elution as positive control
Prep extraction buffer with RNAse inhibitor (1:250) and protease inhibitors (1:100); Add 1:4 (w/v) of extraction buffer to powder
Weigh out 2x 200mg of N2102Ep and 200mg of NTERA
Sonicate samples for 5x2 sec at 2 Amp, repeat once (energy output 100mg samples: ~20J)
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
During spin, wash 40ul of beads per sample (200mg IP) 3x with 1mL of extraction buffer
Set up 200mg IPs with 40ul of beads
1) N2102Ep anti-ORF2
2) NTERA_anti-ORF2
3) N2102Ep_rIgG
IP @ 4°C for 30’
Wash beads 3x 1ml with RNAsin containing wash buffer
Switch beads to fresh tubes at 2nd wash step
Elute in 20ul of LDS @ 70°C for 5’ with mixing; Collect eluate for Western
Add 50mM DTT and 1x LDS (all final concentrations) to each sample
Heat samples @ 70°C for 10’
Split each sample into 2 equal halves (100mg scale), load twice on gel
Load 4-12% Bis-Tris gel;
1) Marker
2) E_N2102Ep anti_ORF2
3) E_NTERA anti_ORF2
4) E_N2102Ep rIgG
5) E_MT302_anti-ORF1 (5% of 100mg IP, used as positive control for ORF2p signal)
6) Space
7) Marker(not shown)
8) Marker
9) E_N2102Ep anti_ORF2
10) E_NTERA anti_ORF2
11) E_N2102Ep rIgG
12) E_MT302_anti-ORF1 (5% of 100mg IP, used as positive control for ORF2p signal)
13) Marker (not shown)
Wet transfer, 70V for 2h
• Cut the membrane between 75 and 50KD, use the top for anti-ORF2 and bottom for anti-ORF1 • Cut top half of the membrane again vertically between lane 6 and 7; probe the left panel with clone 5-5 and right panel with clone 11-1
Block the membranes in TBST/5% milk @ RT, 2hr
Upper panel: Rabbit anti-ORF2 Western
• Left: Primary Ab: Rabbit anti-ORF2 (Clone 5-5, 1.03mg/ml); 1:500, 4°C • Right: Primary Ab: Rabbit anti-ORF2 (Clone 11-1, 0.445mg/ml); 1:300, 4°C
• Secondary Ab: ECL anti-rabbit HRP 1:5,000, RT, 1hr
Lower panel (both lef tand right): Anti-ORF1 Western (Abmart, 4H1, 2mg/ml);
• Primary Ab: Mouse anti-ORF1 antibody, 1:5,000, 4°C, overnight
• Secondary Ab: ECL anti-mouse HRP 1:10,000, RT 1hr
Develop anti-ORF2 Western first; 3 minute exposure, super sensitivity
Put the whole membrane together, short exposure to show ORF1p; 30 second exposure, standard sensitivity, auto tone
