LaCava Research Wiki

Date: 01/08/2018

Cell lines: HEK293 TP53_3xFLAG – transfection for 18hrs HEK293 TP53_3xFLAG – transfection for 36+ hrs

Induction Grow cells in four 75cm² Cell Culture Flasks

When culture reaches >70% confluence, change to induction medium containing Doxycycline (Dox) in concentrations of 0, 1, 5 or 10ng/ml

Make fresh Dox stock (SIGMA D9891)

Dox stock: 5mg/ml = 5000ug/ml

Make 1:500 dilution of the stock (10ul of 5mg/ml Dox + 4990ul of DMEM): 10ug/ml (working stock)

Final concentration of the induction medium: 0, 1ng/ml, 5ng/ml or 10ng/ml

Will need 10ml of induction medium for each cell line

Make media using working stock (10ug/ml)

0 (no Dox)

1ng/ml = 1:10,000 (10ul of 10ug/ml Dox in 50ml of medium)

5ng/ml = 1:2000 (25ul of 10ug/ml Dox in 50ml of medium)

10ng/ml = 1:1000 (50ul of 10ug/ml Dox in 50ml of medium)

Incubate O.N. before harvesting (~20hr induction this time)

Harvesting Remove media and wash the flask with 10ml of DPBS

Add 1.8ml of DPBS; Release the cells using cell scrapers and transfer to a 2ml eppendorf tube with a 2ml serological pipet; Keep on ice

After collecting all samples, spin @ 1k rcf, 4°C for 5’ (Eppendorf Centrifuge 5417R)

Resuspend in 0.5ml extraction buffer with protease inhibitors

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

Sonicate 25x 2 sec @ 2 Amp; Record energy output:

{TP53 (18) 0 = 101J, TP53 1 = 106J, TP53 5 = 99J, TP53 10 = 102J} {TP53 (36) 0 = 101J, TP53 1 = 101J, TP53 5 = 100J, TP53 10 = 103J}

Spin @ 14k rpm, 4°C for 10’ (Eppendorf Centrifuge 5417R)

Transfer the supernatant to fresh tubes (“Sup”)

Resuspend each pellet in 0.5ml of 1.1x LDS by pipetting and vortex; Incubate @70°C for 5’

Spin @ 20k rcf, 25°C for 5’

Keep the supernatant for Western analysis (“Pellet”)

Western Do Bradford Assay of total protein concentration on each sup fraction:

Load 25ug of total protein of each sup fraction and load same vol. of corresponding pellet fraction

Load 25ug of total protein from the supernatant fraction of HEK293 cells (parental cell line); use it as a negative control

Add 50mM DTT (final concentration) to each sample

Heat samples @ 75°C for 10’

Load 25ug of each sample on 15-well 4-12% Bis-Tris gel

26-well Gel:

1. Marker_5ul
2. TP53_18hr_0_Sup
3. TP53_18hr _Dox1_Sup
4. TP53_18hr _Dox5_Sup
5. TP53_18hr _Dox10_Sup
6. TP53_18hr _0_Pellet
7. TP53_18hr _Dox1_Pellet
8. TP53_18hr _Dox5_Pellet
9. TP53_18hr _Dox10_Pellet
10. space
11. TP53_36hr_0_Sup
12. TP53_36hr _Dox1_Sup
13. TP53_36hr _Dox5_Sup
14. TP53_36hr _Dox10_Sup
15. TP53_36hr _0_Pellet
16. TP53_36hr _Dox1_Pellet
17. TP53_36hr _Dox5_Pellet
18. TP53_36hr _Dox10_Pellet
19. space
20. HEK293 cell lysate (TP53 control)
21. LD401_3xFLAG (FLAG control)
22. space
23. Marker_2ul

Wet transfer 70V for 1.5h

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

Primary Ab: mouse anti-TP53 antibody (BioLegend), 1:1000 (4°C overnight)

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

Strip membrane and probe with anti-FLAG antibody 

Anti-FLAG (Sigma F3165) 

Primary Ab: Mouse anti-FLAG antibody, 1:2,000 (4°C overnight)

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