RRP6 purification
Date: 01/29/20
Cell line: HEK-293 EXOSC10-3xFLAG (RRP6-3xFLAG)
Extraction Buffers: 1) 20mM HEPES, pH 7.4, 300mM NaCl, 1% Triton X-100 (v/v)
2) 20mM HEPES, pH 7.4, 100mM MgCl2, 1% Triton X-100 (v/v)
Scale: 250mg with 25ul of anti-FLAG beads
Weigh out 2x 250mg RRP6-3x FLAG; add 1250ul extraction buffer 1 or buffer 2 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; Repeat once (30J total per 250mg sample)
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Take the clarified lysate
Set up 250mg IP reactions (for each buffer condition)
Incubate with 25ul pre-washed beads (10ul beads per 100mg powder)
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 250mg IP, do the following
Add 20ul 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
Add 10ul extraction buffer per tube to wash the beads
Pool these two fractions together (30ul total per 250mg IP)
Pre-equilibrate the Spin X columns with corresponding extraction buffer to avoid volume loss of the samples
Pass the combined fraction through a 0.22um Spin X column
Save 2ul from each sample for gel analysis (Sypro stain)
Snap freeze the samples and keep them in -80C freezer before sending then to NL (dry ice package).
Wash the beads from 250mg IP with 30ul of LDS and load 2ul on gel to see what is left on beads after native elution
Add 1x LDS and 50mM DTT (final concentrations) to gel samples
Heat @ 70°C for 10’
Run samples on 15-well 4-12% Bis-Tris gel for Sypro stain
1) Marker_1ul prestained
2) Space
3) Input RRP6_300mM NaCl (2ul, 6% of 250mg IP)
4) LDS RRP6_300mM NaCl (2ul, 6% of 250mg IP)
5) Space
6) Input RRP6_100mM MgCl2 (2ul, 6% of 250mg IP)
7) Input LDS_100mM MgCl2 (2ul, 6% of 250mg IP)
8) Space
9) BSA_10ng
10) BSA_50ng
11) Space
12) Marker_5ul prestained
Sypro stain, 2 second exposure
