Effects of ZnCl2 and new RNase inhibitor on IP specificity
Date: 5/3/21
Extraction/Wash buffer conditions
Base buffer: 20mM HEPES, pH7.4, 500mM NaCl, 1% Triton X-100 with protease inhibitors with or without ZnCl2 and hUdT
hUdT: 3'-N-hydroxyurea-3'- deoxythymidine 5'-monophosphate, in free form (from Ali Alipour Najmi Iranag, University of Groningen). MW: 380.25g/mole (1M->380.25g/L)
Cell line: LD401 from Sunny (Old LD401 powder with very poor L1 expression)
Scale: 50mg scale with 10ul of mouse IgG beads per reaction
Weigh out 6x 50mg of LD401
Add 200ul of extraction buffer with protease inhibitors and RNase inhibitors to each tube according to 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)
Save 5ul of clarified lysate from 1 (buffer with no RNase); use 1ul for protein assay and the rest for gel
Set up 6x 50mg IP with 10ul mIgG beads per reaction
1. No inhibitor
2. New inhibitor alone
3. 1mM ZnCl2
4. 0.5mM ZnCl2
5. 0.2mM ZnCl2
6. 0.1mM ZnCl2
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) E_no inhibitor
3) E_with inhibitor alone
4) E_1mM Zn
5) E_0.5mM Zn
6) E_0.2mM Zn
7) E_0.1mM Zn
8) Space
9) LD401 lysate_2ug
10) LD401 lysate_5ug
11) Space
12) BSA_50ng
13) BSA_150ng
