Effects of ZnCl2 on IP specificity using CRC tissue
Date: 5/5/21
Extraction/Wash buffer conditions
20mM HEPES, pH7.4, 500mM NaCl, 1% Triton X-100 with protease inhibitors and with no ZnCl2 or 1mM ZnCl2
CRC tissue: 174T
Scale: 50mg scale with 2.5, 5, 10ul of anti-ORF1 or mouse IgG beads per reaction
Weigh out 4x 100mg of 174T
Add 200ul of extraction buffer with protease inhibitors and RNase inhibitors to each tube according to the IP conditions below
Resuspend the powder by vortexing
Sonicate @ 2 Amp; 4x 2sec (15-20J)
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Set up 6x 50mg IP with 10ul anti-ORF1 or mIgG beads per reaction
1. 174T_anti-ORF1_no Zn_5ul beads
2. 174T_mIgG_no Zn_5ul beads
3. 174T_anti-ORF1_0.1mM Zn_2.5ul beads
4. 174T_mIgG_0.1mM Zn_2.5ul beads
5. 174T_anti-ORF1_0.1mM Zn_5ul beads
6. 174T_mIgG_0.1mM Zn_5ul beads
7. 174T_anti-ORF1_0.1mM Zn_10ul beads
8. 174T_mIgG_0.1mM Zn_10ul beads
IP @ 4°C for 30’
During IP, check protein concentration of clarified lysate by Bradford assay
Wash 3x 500ul of extraction buffer
Switch beads to fresh tubes at 2nd wash step
LDS elution: 10ul 1.1x LDS, 70°C for 5’; collect the elution for gel (Coomassie stain)
Protein Gel
Add 1ul of 500mM DTT to each sample
Heat @ 70°C for 10’
Run 2 gels load 50% of elution on each gel (no BSA standards on the gel for Western)
Coomassie gel to check background and Western to check ORF1p yield
Gel 1 for Coomassie stain
1) Marker
2) 174T_anti-ORF1_no Zn_5ul beads
3) 174T_mIgG_no Zn_5ul beads
4) 174T_anti-ORF1_0.1mM Zn_2.5ul beads
5) 174T_mIgG_0.1mM Zn_2.5ul beads
6) 174T_anti-ORF1_0.1mM Zn_5ul beads
7) 174T_mIgG_0.1mM Zn_5ul beads
8) 174T_anti-ORF1_0.1mM Zn_10ul beads
9) 174T_mIgG_0.1mM Zn_10ul beads
10) Space
11) BSA_50ng
12) BSA_150ng
Gel 2 for Western
1) Marker
2) 174T_anti-ORF1_no Zn_5ul beads
3) 174T_mIgG_no Zn_5ul beads
4) 174T_anti-ORF1_0.1mM Zn_2.5ul beads
5) 174T_mIgG_0.1mM Zn_2.5ul beads
6) 174T_anti-ORF1_0.1mM Zn_5ul beads
7) 174T_mIgG_0.1mM Zn_5ul beads
8) 174T_anti-ORF1_0.1mM Zn_10ul beads
9) 174T_mIgG_0.1mM Zn_10ul beads
10) Space
11) MT302 anti-ORF1 (10% of 100mg IP)
Wet transfer 70V for 1.5h
Blocking: TBST/5% milk, RT 2hr
Primary Ab: Mouse anti-ORF1 antibody, 1:5,000, 4°C overnight
Secondary Ab: ECL anti-mouse HRP 1:10,000, RT 1hr
10 seconds exposure, high sensitivity, auto tone
