RRP6 purification with BSA incubation
Date: 11/05/20
Cell line: HEK-293 EXOSC10-3xFLAG (RRP6-3xFLAG) from Leila
Extraction Buffers: 20mM HEPES, pH 7.4, 250mM 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: 50mg with 5ul of anti-FLAG beads (duplicates under each condition)
Weigh out 150mg RRP6-3x FLAG; add 750ul extraction 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 (20J total per 100mg sample)
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Take the clarified lysate
Set up 3x 50mg IP reactions
Incubate with 5ul pre-washed beads per reaction (10ul beads per 100mg powder)
IP @ 4°C for 1h with rotation (cold room)
After 1hr IP, wash beads with 2x 1ml extraction buffer; do the wash in the cold room
Transfer beads to fresh tubes after 2nd wash
Wash beads once with 1ml reaction buffer (see below)
BSA incubation
Spin down briefly and remove any remaining liquid
Add 24ul of reaction buffer with different salt concentration and 0.01% Tx + protease inhibitors1
1) 20mM HEPES, pH 7.4, 250mM NaCl, 0.01%Triton X-100 (v/v)
2) 20mM HEPES, pH 7.4, 200mM NaCl, 0.01%Triton X-100 (v/v)
3) 20mM HEPES, pH 7.4, 100mM NaCl, 0.01%Triton X-100 (v/v)
1ul of 2mg/ml BSA to each tube
4) RRP6_250mM + BSA
5) RRP6_200mM + BSA
6) RRP6_100mM + BSA
Incubate the reaction at RT for 30’ with mixing (1000rpm)
Prepare 1mg/ml 3xFLAG peptide for native elution • Dilute 5mg/ml stock with different reaction buffer with 0.01% Tx
Collect the supernatant after 30’ and save the sample aside (4C)
Load 12.5ul on gel (50% of 50mg reaction; “Sup”)
Wash beads 3x 1ml of extraction buffer
Transfer the beads to fresh tubes during 2nd wash
Native elution
After the 3rd wash, spin down briefly and remove any remaining liquid
Add 10ul 1mg/ml 3xFLAG peptide
Elute for 15 min at RT w/ mixing
Place on a magnet; transfer the supernatant to a fresh tube
Wash the beads with 5ul of reaction buffer
Combine the wash with 3xFLAG elution (15ul total)
Load 7.5ul for gel analysis (“E”; 50% of 50mg IP)
Save 7.5ul for desalting
Wash anti-FLAG beads with 500ul of reaction buffer
Elute beads again with 15ul of LDS, RT for 10’
Load 7.5ul LDS wash on gel (“Beads”; 50% from 50mg IP)
Removing 3x FLAG peptide using Zeba 40K column
Remove the storage buffer from Zeba 40K column, spin @ 1000g for 1’
Equilibrate four 75ul Zeba 40K column (sample range: 3-12ul) with 3x 50ul of 20mM HEPES, pH 7.4, 250, 200 or 100mM NaCl, NO DETERGENT, spin @ 1000g for 1’
Load 7.5ul of sample on column and spin @ 1000g for 2’
Collect the desalted sample for gel analysis (“E-desalted”, 50% of 50mg)
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 Sypro stain
1) Marker_1ul unstained
2) Sup250mM
3) E_250mM
4) E_250mM-desalted
5) Beads_250mM
6) Sup200mM
7) E_200mM
8) E_200mM-desalted
9) Beads_200mM
10) Sup100mM
11) E_100mM
12) E_100mM-desalted
13) Beads_100mM
14) BSA_100ng
15) Marker_5ul prestained
