LaCava Research Wiki

By Nastya and Luciano

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

Extraction/Wash Buffers:

1. 20mM HEPES, pH 7.4, 100mM MgCl2, 1% Triton X-100 (v/v)
2. 20mM HEPES, pH 7.4, 300mM NaCl, 1% Triton X-100 (v/v)

Scale: 100mg with 10ul of anti-FLAG beads

Before beginning, mix extraction buffer aliquots with protease inhibitors; place remaining buffer on ice in preparation to wash beads

Extraction buffers (with protease inhibitors added) should be kept at Room Temp!!

Prepare elution buffers for native elutions – elutions should be done with extraction buffer with 1/10 detergent concentrations (0.1%)

Label with numbers and NaCl concentration then pre-cool 2x 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 2x 100mg RRP6-3x FLAG in 2ml safe-lock eppendorf tubes; add 400ul of appropriate extraction buffer/PI mix (1:4, 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@ 4 Amp, 5x 2 sec; Then repeat 1x (This is the sonication regime we determined was necessary for 700mM NaCl samples (~J total per 100mg sample)

MgCl2 sample of Leila's powder was foaming

Spin @ full speed in benchtop centrifuge (~21k rcf) @ 4°C for 10’ (Eppendorf Centrifuge 5417R)

Wash beads during spin – 2x 10ul of beads; each in a 2ml safe-lock Eppendorf tube, 4x 1ml wash in appropriate extraction/wash buffer (wash buffers do not have protease inhibitor added and should be on ice). After third wash place tubes with beads on ice

Combine supernatants from like buffers; Keep 20ul from each buffer condition (2 total), save at -20C for Western and/or Wes analysis (“SUP”)

Use remaining clarified lysate to set up 2x100mg IP reactions – lysate is added to the tubes of beads and not the other way around Incubate with 10ul pre-washed beads (scale = 10ul beads per 100mg powder)

IP @ 4°C for 1h with rotation (cold room); After IP finishes, heat one thermomixer to 70C, make sure one additional thermomixer is at RT

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. This wash step/transfer is done via micropipette. Removing the beads from one tube and moving to another requires mixing; no vortex/spin is necessary on this wash step.

After removal of the 3rd wash, spin beads down briefly on benchtop minifuge and remove any remaining liquid, usually ~1-2ul

Add 10ul 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 20ul 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 100mg IP

Save 15ul 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 with shaking, then collect final eluate fraction. Use 15ul of this fraction for gel

Add LDS and 50mM DTT (final concentrations) to gel samples

Heat samples @ 70°C for 10’ to denature

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

1. Ladder
2. Hua powder MgCl2 native
3. Hua powder MgCl2 LDS
4. Hua powder NaCl native
5. Hua powder NaCl LDS
6. ERIBA powder MgCl2 native
7. ERIBA powder MgCl2 LDS
8. ERIBA powder NaCl native
9. ERIBA powder NaCl LDS
10. BSA 200ng

WB with MgCl2 to test new ZCCHC8 Ab 1:1000

1 ul of SUP / Ft / n.el

9,5 ul of LDS el.