RRP6 purification with 300mM and 500mM buffer
Date: 09/17/20
Cell line: HEK-293 EXOSC10-3xFLAG (RRP6-3xFLAG) from Leila
Extraction Buffers:
1) 20mM HEPES, pH 7.4, 300mM NaCl, 1% Triton X-100 (v/v)
2) 20mM HEPES, pH 7.4, 500mM 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
Scale: 50mg with 5ul of anti-FLAG beads
Weigh out 2x100mg RRP6-3x FLAG; add 1000ul 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 (20J total per 100mg sample)
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Take the clarified lysate
Set up 4x 50mg IP reactions (2 for each buffer condition)
Incubate with 5ul pre-washed beads (10ul beads per 100mg powder)
IP @ 4°C for 1h with rotation (cold room)
Prepare 1mg/ml 3xFLAG peptide for native elution (10ul per 50mg reaction)
1) Dilute 5mg/ml stock with 300mM extraction buffer (10ul)
2) Dilute 5mg/ml stock with 300mM extraction buffer with 0.01% Tx) (2x10ul)
3) Dilute 5mg/ml stock with 500mM extraction buffer (10ul)
After 1hr IP, wash beads with 2x 1ml extraction buffer; do all wash steps in the cold room
Transfer the beads to fresh tubes during 2nd wash
Take one tube from each buffer condition (either 300mM or 500mM salt), wash with 300mM buffer with 0.01% Tx; followed by elution with 1mg/ml 3x FLAG with 0.01% detergent
1) 300mM extraction_300mM wash with 1% Tx_300mM elution+1% Tx
2) 300mM extraction_300mM wash with 0.01% Tx _300mM elution+0.01% Tx
3) 500mM extraction_500mM wash with 1% Tx_500mM elution+0.01% Tx
4) 500mM extraction_300mM wash with 0.01% Tx _300m M elution+0.01% Tx
After the 3rd wash, spin down briefly and remove any remaining liquid
Add 10ul 1mg/ml 3xFLAG peptide in extraction buffer to each tube based on the description above
Elute for 15 min at RT w/ mixing
Place on a magnet; transfer the supernatant to a fresh tube
Wash the beads with 10ul at RT for 10’ followed by 2nd LDS wash @ 70°C for 5’
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) 1a_FLAG elution
3) 1b_LDS_RT
4) 1c_LDS_70C
5) 2a_FLAG elution
6) 2b_LDS_RT
7) 2c_LDS_70C
8) 3a_FLAG elution
9) 3b_LDS_RT
10) 3c_LDS_70C
11) 4a_FLAG elution
12) 4b_LDS_RT
13) 4c_LDS_70C
14) BSA_100ng
15) Marker_5ul prestained
IP Conditions
1) 300mM extraction_300mM wash with 1% Tx_300mM elution+1% Tx
2) 300mM extraction_300mM wash with 0.01% Tx _300mM elution+0.01% Tx
3) 500mM extraction_500mM wash with 1% Tx_500mM elution+0.01% Tx
4) 500mM extraction_300mM wash with 0.01% Tx _300m M elution+0.01% Tx
Conclusion:
Regarding Exosome IP: 300mM extraction buffer gave better yield of SKIV2L2; salt and detergent concentrations in last wash and elution buffer did not affect the yield
Regarding anti-FLAG beads: 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. We did not observe IgG leakage during native elution with 3x FLAG peptide @ RT. Elution with LDS @ RT washed off most protein bound to anti-FLAG beads, but not anti-FLAG antibody. 2nd LDS elution @ 70°C removed IgG from beads with minor gain of target protein, indicating we could do LDS elution @ RT for anti-FLAG beads avoiding IgG leakage problem.