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Cell line: HEK-293 EXOSC10-3xFLAG (RRP6-3xFLAG)

Four conditions; three replicates per condition (one set of conditions each performed once by Leila, Arianna, Mehrnoosh)

Extraction/Wash Buffers:

1. 20mM HEPES, pH 7.4, 300mM NaCl, 1% Triton X-100 (v/v)
2. 20mM HEPES, pH 7.4, 600mM NaCl, 1% Triton X-100 (v/v)
3. 20mM HEPES, pH 7.4, 650mM NaCl, 1% Triton X-100 (v/v)
4. 20mM HEPES, pH 7.4, 700mM 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 -80C 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) @ 4C 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 @ 4C 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 ~1-2ul

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 without detergent 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 and to remove any wetting agents from the membrane (add 100ul of corresponding extraction buffer without 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 @ 70C for 10’ to denature

Run samples on 2x 15-well 4-12% Bis-Tris gel for Blue-Silver stain

Gel #1

1. Marker_5ul prestained
2. Native RRP6_300mM NaCl (10ul, 50% of 250mg IP)Leila
3. LDS RRP6_300mM NaCl (10ul, 50% of 250mg IP)
4. Native RRP6_600mM NaCl (10ul, 50% of 250mg IP)Leila
5. LDS RRP6_600mM NaCl (10ul, 50% of 250mg IP)
6. Native RRP6_300mM NaCl (10ul, 50% of 250mg IP)Arianna
7. LDS RRP6_300mM NaCl (10ul, 50% of 250mg IP)
8. Native RRP6_600mM NaCl (10ul, 50% of 250mg IP)Arianna
9. LDS RRP6_600mM NaCl (10ul, 50% of 250mg IP)
10. Native RRP6_300mM NaCl (10ul, 50% of 250mg IP)Mehrnoosh
11. LDS RRP6_300mM NaCl (10ul, 50% of 250mg IP)
12. Native RRP6_600mM NaCl (10ul, 50% of 250mg IP)Mehrnoosh
13. LDS RRP6_600mM NaCl (10ul, 50% of 250mg IP)
14. BSA_10ng
15. BSA_50ng

Gel #2

1. Marker_5ul prestained
2. Native RRP6_650mM NaCl (10ul, 50% of 250mg IP)Leila
3. LDS RRP6_650mM NaCl (10ul, 50% of 250mg IP)
4. Native RRP6_700mM NaCl (10ul, 50% of 250mg IP)Leila
5. LDS RRP6_700mM NaCl (10ul, 50% of 250mg IP)
6. Native RRP6_650mM NaCl (10ul, 50% of 250mg IP)Arianna
7. LDS RRP6_650mM NaCl (10ul, 50% of 250mg IP)
8. Native RRP6_700mM NaCl (10ul, 50% of 250mg IP)Arianna
9. LDS RRP6_700mM NaCl (10ul, 50% of 250mg IP)
10. Native RRP6_650mM NaCl (10ul, 50% of 250mg IP)Mehrnoosh
11. LDS RRP6_650mM NaCl (10ul, 50% of 250mg IP)
12. Native RRP6_700mM NaCl (10ul, 50% of 250mg IP)Mehrnoosh
13. LDS RRP6_700mM NaCl (10ul, 50% of 250mg IP)
14. BSA_10ng
15. BSA_50ng