LaCava Research Wiki

Date: 01/24/2018

Cell lines:
HEK293 FLp-In T-Rex (4/5/17, control) HEK293 TP53-3xFLAG (1/23/18)

• Extraction buffers:
  20mM HEPES, pH7.4, 0.5% Tritonx-100, 150mM NaCl
• 20mM HEPES, pH7.4, 0.5% Tritonx-100, 300mM NaCl

Weigh out 100mg of HEK293 control and 2x 100mg of HEK293 TP53-3xFLAG; add 400ul of extraction buffer to each tube (1:4 w/v), vortex to mix

Sonicate 25x 2 sec at 2 Amp (Energy output ~100J)  

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

Keep the supernatant, split each into 2 equal parts, set up the following 50mg IP reactions with anti-FLAG beads per reaction:

• 5ul of anti-FLAG beads (duplicates 1a, 1b) • 5ul of anti-FLAG beads (duplicates 2a, 2b) • TP53_5ul of anti-FLAG beads (duplicates 3a, 3b)

Incubate @ 4°C for 30’

After IP, wash 3x 1ml extraction buffer
  Switch beads to fresh tubes at 2nd wash step

Elute with either 15ul of LDS (70°C for 5’) or 10ul of 1mg/ml 3x FLAG (RT for 15’)

1. HEK_2.5ul beads_LDS
2. HEK_2.5ul beads_3xFLAG
3. TP53_2.5ul beads_LDS
4. TP53_2.5ul beads_3xFLAG
5. TP53_5ul beads_LDS
6. TP53_5ul beads_3xFLAG

Add 3.5ul of 4x LDS to 3x FLAG elution

Add DTT to 50mM to all samples and heat @75 °C for 10’
  Run 15-well 4-12% Bis-Tris gel for Coomassie stain

1. space
2. Marker
3. space
4. HEK_2.5ul beads_LDS
5. HEK_2.5ul beads_3xFLAG
6. space
7. TP53_2.5ul beads_LDS
8. TP53_2.5ul beads_3xFLAG
9. TP53_5ul beads_LDS
10. TP53_5ul beads_3xFLAG
11. space
12. BSA_50ng
13. BSA_150ng