RRP6 purification
Date: 04/12/21
Cell line: HEK-293 EXOSC10-3xFLAG (RRP6-3xFLAG)
Extraction Buffer: 20mM HEPES, pH 7.4, 300mM NaCl, 1% Triton X-100 (v/v)
Scale: 100mg with 10ul of anti-FLAG beads
Weigh out 100mg RRP6-3x FLAG (old 7/2/20; new 9/4/21); add 500ul extraction buffer with protease inhibitors to each (1:5, w/v) to each tube
Vortex to mix; put the tubes on ice after resuspending the powder
Sonicate @ 2 Amp, 5x 2 sec;
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Take the clarified lysate
Set up 100mg IP reactions
Incubate with 10ul pre-washed beads
IP @ 4°C for 1h with rotation (cold room)
Wash beads with 3x 1ml extraction buffer; do all wash steps in the cold room
Transfer the beads to fresh tubes during 2nd wash
After the 3rd wash, spin down briefly and remove any remaining liquid
For one of the 100mg IP, do the following
Add 10ul 1mg/ml 3xFLAG peptide in extraction buffer to each tube (1mg/ml 3x FLAG peptide was diluted from 5mg/ml stock in extraction buffer)
Elute for 15 min at RT w/ mixing
Place on a magnet; transfer the supernatant to a fresh tube
Wash the beads with 10ul of 1.1xLDS (RT for 10’ with mixing) to see what is left on beads after native elution
Add LDS to 3x FLAG elution and 50mM DTT (final concentrations) to all gel samples
Heat @ 70°C for 10’
Run samples on 15-well 4-12% Bis-Tris gel for Aquastain
1) Marker
2) RRP6_7/2/21_3xFLAG
3) RRP6_7/2/21_LDS
4) RRP6_9/4/21_3xFLAG
5) RRP6_9/4/21_LDS
6) BSA_50ng
7) BSA_150ng
