N2102Ep anti-ORF1 and anti-ORF2 IPs with controls
Date: 10/12/20
• RNAse free L1 extraction buffer. 1:250 RNasin in EB, 1:1000 in wash.
• 100mg scale with 20ul beads
• Cell lines: N2102Ep (4x100mg) and MT302 (positive control, 100mg)
1. Prep extraction buffer with RNAse inhibitor (1:250) and protease inhibitors (1:100); Add 1:4 (w/v) of extraction buffer to powder
2. Sonicate samples for 5x2 sec at 2 Amp (energy output 100mg samples: ~20J)
3. Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R); save 10ul of input
4. During spin, wash 20ul of beads per sample with extraction buffer that does NOT have RNasin added (to conserve RNasin). (Beads stored in 1X PBS, 0.5ng/uL BSA, 50% glycerol.)
To wash beads, add 1mL wash buffer to each tube first, then add 20uL of bead slurry to each buffer-filled tube. Wash 3x with 1mL
6. Combined clarified lysate from 4x 100mg N2102Ep, sake 20ul for Western (“Input”) Use the rest to set up 4x 100mg IPs with 20ul beads per 100mg reaction; MT302 lysate was used as positive control
1) N2102Ep anti-ORF1
2) N2102Ep_mIgG
3) N2102Ep anti-ORF2
4) N2102Ep_rIgG
5) MT302_anti-ORF1
7. IP @ 4°C for 30’
8. Save 10ul of FT after IP
9. Wash beads 3x 1ml with RNAsin containing wash buffer
10. Switch beads to fresh tubes at 2nd wash step
11. Elute in 15ul of LDS @ 70°C for 5’ with mixing
12. Collect eluate for Western
Determine total protein concentration of input by Bradford Assay (12.56.ug/ul); load 25ug on gel (2ul); load same volume of FT on gel
Add 50mM DTT and 1x LDS (all final concentrations) to each sample
Heat samples @ 70°C for 10’
Load 4-12% Bis-Tris gel
1) Marker
2) Input
3) FT-anti-ORF2
4) FT_rIgG
5) E_N2102Ep_anti-ORF2
6) E_2102Ep_rIgG
7) Space
8) E_N2102Ep_anti-ORF1 (5% of 100mg IP, used as positive control for both ORF1p and ORF2p signals)
9) E_MT302_anti-ORF1 (5% of 100mg IP, used as positive control for ORF2p signal)
10) Space
11) Marker
12) E_N2102Ep_anti-ORF1 (95% of 100mg IP, trying to detect ORF2 signal; ORF1 signal will be too strong for Western)
13) E_2102Ep_mIgG
14) E_MT302_anti-ORF1 (5% of 100mg IP, used as positive control for ORF2p signal)
15) Marker (not shown)
Wet transfer, 70V for 2h
• Cut the membrane first vertically between lane 10 and 11 before the 2nd Marker; use the right panel for anti-ORF2 Western
• Cut the left panel membrane again between 75 and 50KD, use the top for anti-ORF2 and bottom for anti-ORF1
Block the membranes in TBST/5% milk @ RT, 2hr
Upper left panel and right panel: Rabbit anti-ORF2 Western (Clone 5-5, 1.03mg/ml);
• Primary Ab: Rabbit anti-ORF2 antibody, 1:500, 4°C, overnight (upper left panel: antibody that has been used once before; right panel: fresh antibody)
• Secondary Ab: ECL anti-rabbit HRP 1:10,000, RT, 1hr
Lower panel: Anti-ORF1 Western (Abmart, 4H1, 2mg/ml);
• Primary Ab: Mouse anti-ORF1 antibody, 1:5,000, 4°C, overnight; used old primary antibody trying to reduce background
• Secondary Ab: ECL anti-mouse HRP 1:10,000, RT 1hr
Left panel: E2102Ep anti-ORF2 IP_100mg
10 second exposure; high sensitivity, auto tone
Upper left panel_first long exposure:
5 minute exposure, super sensitivity, auto tone
Rinse the membrane with TBST for 10’
Incubate with secondary antibody 1:5,000 instead of 1:10,000 for 30’
3 quick rinse followed by 3x 10’ wash with TBST
Redevelop Western
1 minute exposure, high sensitivity, auto tone
2 minute exposure, super sensitivity, no adjustment
Right panel: E2102Ep anti-ORF1 IP_100mg
1 minute exposure, super sensitivity, auto tone
Strip the membrane and reprobe it with anti-ORF2, clone 9-7 (1.39mg/ml; 1;1000); 4°C, overnight
4 minute exposure, super sensitivity, no auto tone
