STAT3-3xFLAG PO comparing anti-FLAG beads and anti-STAT3 beads
Date: 08/0717
Cell line: HEK293 STAT3-3xFLAG (6/30/17)
Extraction buffer: 20mM HEPES, pH7.4, 0.5% Tritonx-100, 300mM NaCl
Weigh out 2x 100mg of HEK293 STAT3-3xFLAG; add 400ul of extraction buffer to each tube (1:4 w/v), vortex to mix
Sonicate 5x 2 sec at 2 Amp (Energy output ~20J)
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Combine the supernatant and then split into 2 equal parts, set up the following 50mg IP reactions with anti-FLAG beads per reaction:
STAT3_5ul of anti-STAT3 beads_032717 STAT3_5ul of anti-FLAG beads_030917
Incubate @ 4°C for 30’
After IP, wash 3x 1ml extraction buffer
Switch beads to fresh tubes at 2nd wash step
Elute anti-STAT3 beads with either 15ul of LDS (70°C for 5’)
Elute anti-FLAG beads first with 10ul of 1mg/ml 3x FLAG (RT for 15’), followed by 15ul of LDS (70°C for 5’)
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
- Marker
- LDS_anti-STAT3 beads
- 3xFLAG elution_anti-FLAG beads
- LDS_anti-FLAG beads
- BSA_50ng
- BSA_150ng
