Effects of ZnCl2 on IP specificity using different cell lines and tissue
Date: 5/4/21
Extraction/Wash buffer conditions
20mM HEPES, pH7.4, 500mM NaCl, 1% Triton X-100 with protease inhibitors and different concentrations of ZnCl2
Cell lines and tissue:
LD401 from Sunny (Old LD401 powder with very poor L1 expression): to test Zn titer in mIgG IP N2102Ep, NTREA and 174T (tumor tissue): to compare anti-ORF1 and mIgG IP
Scale: 50mg scale with 10ul of anti-ORF1 or mouse IgG beads per reaction
Weigh out 4x 50mg of LD401 and 2x50mg of other cell lines and tissue
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 mIgG beads per reaction
1. LD401_mIgG_no Zn
2. LD401_mIgG_0.025mM Zn
3. LD401_mIgG_0.05mM Zn
4. LD401_mIgG_0.1mM Zn
5. N2102Ep_anti-ORF1_0.1mM Zn
6. N2102Ep_mIgG_0.1mM Zn
7. NTERA_anti-ORF1_0.1mM Zn
8. NTERA_mIgG_0.1mM Zn
9. 174T_anti-ORF1_0.1mM Zn
10. 174T_mIgG_0.1mM Zn
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 4-12% Bis-Tris gel
1) Marker
2) LD401_mIgG_no Zn
3) LD401_mIgG_0.025mM Zn
4) LD401_mIgG_0.05mM Zn
5) LD401_mIgG_0.1mM Zn
6) Space
7) N2102Ep_anti-ORF1_0.1mM Zn
8) N2102Ep_mIgG_0.1mM Zn
9) NTERA_anti-ORF1_0.1mM Zn
10) NTERA_mIgG_0.1mM Zn
11) 174T_anti-ORF1_0.1mM Zn
12) 174T_mIgG_0.1mM Zn
13) Space
14) BSA_50ng
15) BSA_150ng
