RRP6 purification followed by dialysis
Date: 05/14/21
Cell line: HEK-293 EXOSC10-3xFLAG (RRP6-3xFLAG) from Leila
Extraction Buffers: 20mM HEPES, pH 7.4, 300mM NaCl, 1% Triton X-100 (v/v)
Anti-FLAG beads used in this experiment were made with anti-FLAG antibody from Marty (beads made on 09/03/20) with IgG leakage problem when elute with LDS @ 70°C
IP
Scale: 100mg with 10ul of anti-FLAG
Weigh out 2x 100mg RRP6-3x FLAG; add 500ul extraction with protease inhibitors to (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 (20J total per 100mg sample)
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Take the clarified lysate
Set up 100mg IP reactions
Incubate with 10ul pre-washed anti-FLAG beads
IP @ 4°C for 1h with rotation (cold room)
Prepare 1mg/ml 3xFLAG peptide for native elution
• Dilute 5mg/ml stock with extraction buffer with 0.01% Tx
After 1hr IP, wash beads with 3x 1ml extraction buffer; do the wash in the cold room
Transfer beads to fresh tubes after 2nd wash
Native elution
After the 3rd wash, spin down briefly and remove any remaining liquid
Add 10ul 1mg/ml 3xFLAG peptide per tube (100mg IP)
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 reaction buffer
Combine the wash with 3xFLAG elution from both tubes (40ul total)
Save 10ul for gel (50mg equivalent)
Save 30ul for dialysis
Wash anti-FLAG beads with 500ul of reaction buffer
Elute beads again with 40ul of LDS, RT for 10’
Load 10ul LDS wash on gel (“Beads”; 25mg equivalent)
Removing 3x FLAG peptide using Slide-A-Lyzer MINI Dialysis Device 7K (ThermoFisher, cat # 69560)
Prepare dialysate (1L): 20mM HEPES, pH 7.4, 300mM NaCl, NO DETERGENT
Place the unit into the float so that the bottom of the dialysis unit is in contact with the dialysate. Always make sure that the volume level of the sample is at or above the level of the dialysate.
Use two units, load 20ul sample to one and 10ul to the other one (unit capacity is 500ul; for best result, load 10-100ul of sample)
Cap the unit and place in a flotation device (can use parafilm instead of the cap)
Use a low-speed setting on a stir plate so that the flotation device is not submerged
Dialyze for 1-2 hr using a dialysate volume of 1L
After 1hr, take 10ul out from unit 1 (initial volume loaded 20ul) and put the unit back on the floater to dialyze for another hour
After 2hr, take both units out and collect the samples
Load 10ul of each sample on gel (50mg equivalent); save the rest @ 4°C (~10ul after 2hr dialysis)
Add LDS to peptide 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 Coomassie stain
1) Marker_5ul prestained
2) LDS_anti-FLAG beads
3) Space
4) Input_10ul 3xFLAG elution before dialysis
5) Dialyzed sample 1_10ul after 1hr dialysis
6) Dialyzed sample 2_10ul after 2hr dialysis
7) BSA_50ng
8) BSA_150ng
