LaCava Research Wiki

Test PO with anti-TP53 beads

Date: 08/09/17

Cell lines and tissue:

HEK293 FLp-In T-Rex (4/5/17)
HeLa Kyoto (8/11/16)
163T (2015-P-1858, Colon Tumor)

Beads were conjugated with anti-TP53 (3/16/17)

Scale: 100mg per experiment, with 10ul of beads

Extraction/wash buffers:

1)    20mM HEPES, pH7.4, 0.5% Tritonx-100, 150mM NaCl
2)    20mM HEPES, pH7.4, 0.5% Tritonx-100, 300mM NaCl

Weigh out 2x 100mg of each powder;

Add 400ul of either extraction buffer 1) or 2) with protease inhibitors to each tube (1:4 w/v), vortex to mix

Sonicate (25x 2 sec) @ 2 Amp

Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)

Save 15ul of the supernatant for Western analysis (“Sup”)

Resuspend each pellet in 450ul of 1.1x LDS by pipetting and vortex

Spin @ 20k rcf, 4°C for 5’

Keep the supernatant for Western analysis (“Pellet”)

Use the rest of the clarified lysate to set up IPs using 10ul of beads for 100mg of powder

1. HEK_150mM NaCl
2. HEK_300mM NaCl
3. HeLa_150mM NaCl
4. HeLa_300mM NaCl
5. Colon Tumor_150mM NaCl
6. Colon Tumor_300mM NaCl

Incubate @ 4°C for 30’

Keep the flow-through for Western analysis (“FT”)

Wash 3x 1ml extraction buffer (transfer the beads to fresh tubes during 2nd wash)

Elute with 18ul of LDS @ 70°C for 5’ with shaking

Collect the elution (“E”)

Add 2ul of 500mM DTT to each sample (total 20ul)

Heat samples @ 75°C for 10’

Load 90% of the elution for Coomassie stain and use 10% for Western

Run 15-well 4-12% Bis-Tris gel, stain with Coomassie

1. Marker
2. HEK_150mM NaCl
3. HEK_300mM NaCl
4. HeLa_150mM NaCl
5. HeLa_300mM NaCl
6. 163CT_150mM
7. 163CT_300mM
8. BSA_50ng
9. BSA_150ng

Western of Sup, Pellet, FT and E

26-well 4-12% Bis-Tris gel (Sup, Pellet, FT and E; 2ul for each fraction)

1. Marker
2. Sup_HEK_150
3. Sup_HEK_300
4. FT_HEK_150
5. FT_HEK_300
6. Pellet_HEK_150
7. Pellet_HEK_300
8. Sup_HeLa_150
9. Sup_HeLa_300
10. FT_HeLa_150
11. FT_HeLa_300
12. Pellet_HeLa_150
13. Pellet_HeLa_300
14. Sup_CT_150
15. Sup_CT_300
16. FT_CT_150
17. FT_CT_300
18. Pellet_CT_150
19. Pellet_CT_300
20. E_HEK_150
21. E_HEK_300
22. E_HeLa_150
23. E_HeLa_300
24. E_CT_150
25. E_CT_300
26. Marker

Wet transfer, 70V for 1.5hr

Block in TBST/5%milk @ RT for 1hr

Anti-TP53

Primary Ab: mouse anti-TP53 antibody, 1:1,000 ( 4°C overnight)

Secondary Ab: HRP linked anti-mouse IgG, 1:10,000 (RT for 1hr)

2-min exposure, high sensitivity (Elutions last lanes)

10-min exposure, high sensitivity (Elutions last lanes)