IMMUNOPRECIPITATION FOR EXOSC-10-3XFLAG PURIFICATION
For the cryomilling procedure I followed this protocol by LaCava et. al. (https://www.jove.com/pdf/54518/jove-protocol-54518-protein-complex-affinity-capture-from-cryomilled-mammalian-cells)
IMMUNOPRECIPITATION of HEK-293 EXOSC10-3xFLAG
Cell line: HEK-293 EXOSC10-3xFLAG (RRP6-3xFLAG)
Aim: The objective is to purify and to affinity capture EXOSC10-3xFLAG from HEK-293 cell line, specifically I tested four different conditions at increasing [NaCl] in non-denaturing (- LDS) and denaturing (+ LDS) conditions, so that progressively the main subunits of the main complex should detach; I performed three replicates per condition
Materials: - Liquid nitrogen - Protease inhibitors - 20mM HEPES (previously filtered with 0,22 µm filters) - 300, 600, 650, 700 mM NaCl - 1% Triton X-100 (v/v) - Epoxy beads - 3x FLAG-peptide - Spin-X columns
Methods: Extraction/Wash Buffers: - 20mM HEPES, pH 7.4, 300 mM NaCl, 1% Triton X-100 (v/v) - 20mM HEPES, pH 7.4, 600 mM NaCl, 1% Triton X-100 (v/v) - 20mM HEPES, pH 7.4, 650 mM NaCl, 1% Triton X-100 (v/v) - 20mM HEPES, pH 7.4, 700 mM NaCl, 1% Triton X-100 (v/v)
- Scale: 250mg with 25ul of anti-FLAG beads
- Before beginning, mix extraction buffer aliquots with protease inhibitors; place remaining buffer on ice in preparation to wash beads
- Prepare elution buffers for native elutions – elutions should be done with extraction buffer with 1/10 detergent concentrations (0.1%)
- Pre-cool 2ml safe-lock Eppendorf tubes in LN2; also pre-cool weighing tools and large tweezers
- Take powder from -80ºC and place in liquid nitrogen
- Weigh out 4x 250mg RRP6-3x FLAG in 2ml safe-lock eppendorf tubes; add 1250ul of appropriate extraction buffer/PI mix (1:5, w/v) to each tube
- Vortex at full speed to mix; put the tubes on ice after resuspending the powder. If 10 seconds of vortexing at full speed does not resuspend powder, place tube on ice for 10 seconds before vortexing again. Repeat until powder is completely resuspended or until only small fluffy aggregates remain in the tube
- Sonicate in cold room @2 Amp, 5x 2 sec; Repeat once (~30J total per 250mg sample)
- Spin @ full speed in benchtop centrifuge (~21k rcf) @ 4ºC for 10’ (Eppendorf Centrifuge 5417R)
- Wash beads during spin
- Keep 20ul from each tube, save at -20C for Western and/or Wes analysis (“SUP”)
- Use remaining clarified lysate to set up 4x 250mg IP reactions
- Incubate with 25ul pre-washed beads (scale = 10ul beads per 100mg powder)
- IP @ 4ºC for 1h with rotation (cold room)
- After IP, vortex at 5-7 setting if necessary; brief spin in benchtop minifuge; place beads on magnet and save 20ul of FT for Western. Discard remaining FT
- Wash beads with 3x 1ml extraction buffer; do all wash steps in the cold room/on ice
- Transfer the beads to fresh tubes during 2nd wash to prevent carryover of anything from IP into elution
- After the 3rd wash, spin down briefly on benchtop minifuge and remove any remaining liquid, usually ~1-2 µL
- Add 20ul 1mg/ml 3xFLAG peptide in extraction buffer to each tube (1mg/ml 3x FLAG peptide was diluted from 4mg/ml stock in extraction buffer with reduced detergent concentration)
- Elute for 15 min at RT w/ mixing in Eppendorf Thermomixer
- Place on a magnet; collect the supernatant (native eluate fraction) in a fresh tube
- Add 10ul extraction buffer per tube to wash the beads; collect this fraction and add to the native eluate fraction. Pooled fractions together should be 30ul total per 250mg IP
- Pre-equilibrate the Spin X columns with corresponding extraction buffer to avoid volume loss of the samples (add 100ul of corresponding extraction buffer with reduced detergent to column then spin @ 16000 rcf for 1 minute)
- Pass the combined fraction through a 0.22um Spin X column
- Save 10ul from each sample for Blue-Silver gel analysis
- Add 30ul of 1.1x LDS to beads to recover any remaining protein; heat @ 70C for 5 minutes, then collect final eluate fraction. Use 10ul of this fraction for gel
- Add LDS and 50mM DTT (final concentrations) to gel samples - Heat samples @ 70ºC for 10’ to denature - Run samples on 2x 15-well 4-12% Bis-Tris gel for Blue-Silver stain
Results: The gel was set up as it follows: 1) Marker_5ul prestained 2) Native RRP6_300mM NaCl (10ul, 50% of 250mg IP) 3) LDS RRP6_300mM NaCl (10ul, 50% of 250mg IP) 4) Native RRP6_600mM NaCl (10ul, 50% of 250mg IP) 5) LDS RRP6_600mM NaCl (10ul, 50% of 250mg IP) 6) Native RRP6_300mM NaCl (10ul, 50% of 250mg IP) 7) LDS RRP6_300mM NaCl (10ul, 50% of 250mg IP) 8) Native RRP6_600mM NaCl (10ul, 50% of 250mg IP) 9) LDS RRP6_600mM NaCl (10ul, 50% of 250mg IP) 10) Native RRP6_300mM NaCl (10ul, 50% of 250mg IP) 11) LDS RRP6_300mM NaCl (10ul, 50% of 250mg IP) 12) Native RRP6_600mM NaCl (10ul, 50% of 250mg IP) 13) LDS RRP6_600mM NaCl (10ul, 50% of 250mg IP) 14) BSA_10ng 15) BSA_50ng
At increasing [NaCl], as hypnotized, I noticed a slightly visible loss of MTREX cofactor.
As the detergent seemed to interfere with MP measurements I performed the same IP decreasing the concentration tenfold (from 1% Triton X-100 to 0.1% Triton X-100).