LaCava Research Wiki

Cell line: HEK293 FLp-In T-Rex (23/5/18)

Scale: 100mg per experiment

Extraction/wash buffer:
20mM HEPES, pH7.4, 0.5% Tritonx-100, 300mM NaCl

Label all tubes

Weigh out 8x 100mg of powder;

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

Sonication conditions:

1. 300mM NaCl_5x 2 sec @ 2 Amp (with 1sec pause); energy output J
2. 300mM NaCl_5x 2 sec @ 4 Amp (with 1sec pause); energy output J
3. 300mM NaCl_25x 2 sec @ 2 Amp (with 1sec pause); energy output J
4. 300mM NaCl_25x 2 sec @ 4 Amp (with 1sec pause); energy output J
5. 300mM NaCl_50x 2 sec @ 2 Amp (with 1sec pause); energy output J
6. 300mM NaCl_50x 2 sec @ 4 Amp (with 1sec pause); energy output J
7. 300mM NaCl_100x 2 sec @ 2 Amp (with 1sec pause); energy output J
8. 300mM NaCl_100x 2 sec @ 4 Amp (with 1sec pause); energy output J

Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R); Save the supernatant for Western analysis (“Sup”)

Resuspend each pellet in 450ul of 1.1x LDS by pipetting and vortex; Incubate @95°C for 5’

Spin @ 20k rcf, 25°C for 5’; Keep the supernatant for Western analysis (“Pellet”)

Do Bradford Assay of total protein concentration on each sup fraction to determine WB loading amounts

Load 25ug of total protein of each sup fraction and load same vol. of corresponding pellet fraction (total protein conc for these samples:

Add 2ul of 500mM DTT to each sample

Heat samples @ 75°C for 10’

Run Sup and Pellet on 26-well 4-12% Bis-Tris gel for Western for TP53

1. Marker
2. SUP_5x2_2 Amp
3. SUP_5x2_4 Amp
4. SUP_25x2_2 Amp
5. SUP_25x2_4 Amp
6. SUP_50x2_2 Amp
7. SUP_50x2_4 Amp
8. SUP_100x2_2 Amp
9. SUP_100x2_4 Amp
10. Pellet_5x2_2 Amp
11. Pellet _5x2_4 Amp
12. Pellet _25x2_2 Amp
13. Pellet _25x2_4 Amp
14. Pellet _50x2_2 Amp
15. Pellet _50x2_4 Amp
16. Pellet _100x2_2 Amp
17. Pellet _100x2_4 Amp
18. Space
19. BSA_50ng
20. BSA_150ng
21. Marker

Wet transfer, 70V for 1.5hr

Block in TBST/5% Milk @ RT for 2hr

Primary Ab: mouse anti-TP53 antibody (BioLegend, Cat # 645702), 1:1,000 ( 4°C overnight)

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

WES results from 26.05.20