Cell line: HEK-293 EXOSC10-3xFLAG (RRP6-3xFLAG)
Extraction/Wash Buffers:
- 20mM HEPES, pH 7.4, 300mM NaCl, 1% Triton X-100 (v/v); in triplicate
- 20mM HEPES, pH 7.4, 700mM NaCl, 1% Triton X-100 (v/v); in triplicate
Scale: 125mg with 12.5ul of anti-FLAG beads per sample
Before beginning:
- place sonicator probe in cold room
- 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%)
This approach is being taken to align the conditions used for MP and MS
Label with numbers and NaCl concentration then pre-cool 6x 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 6x 125mg RRP6-3x FLAG in 2ml safe-lock eppendorf tubes; add 625ul 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 700mM salt samples first; testing for viscosity. Same regime should be applied to all samples.
Sonicate in cold room@ 4 Amp, 5x 2 sec; (~_ total per 125mg sample - record new J values); after round one of sonication, check for viscosity by pipetting. If sample is viscous, repeat sonication, then check again.
Spin @ full speed in benchtop centrifuge (~21k rcf) @ 4°C for 10’ (Eppendorf Centrifuge 5417R); save 20ul for Western analysis (“SUP”); place on ice or at -20C
Wash beads during spin –3x 12.5mg of beads; each in a 2ml safe-lock Eppendorf tube, 3x 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
Use remaining clarified lysate to set up 6x 125mg IP reactions – lysate is added to the tubes of beads and not the other way around
Incubate with 12.5ul 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, 1 part of (4 mg/ml) peptide to 4 parts of buffer, total 5 parts. Means 5 ul peptide in 20 ul of buffer, will be 25 ul total)
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 125mg IP
Save 6ul from each eluate for WB, Wes and 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 10ul of this fraction for gel
Add 4x 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
- Marker_5ul prestained
- Native RRP6_300mM NaCl (Pooled replicates)
- LDS RRP6_300mM NaCl (Pooled replicates)
- Native RRP6_700mM NaCl (Pooled replicates)
- LDS RRP6_700mM NaCl (Pooled replicates)
- space
- BSA_50ng
- BSA_250ng
- BSA_500ng
For Western, 26-well 4-12% Bis-Tris gel, run gel all the way out to allow separation of flag tagged and endogenous protein:
- Marker 5ul
- 300mM NaCl SUP
- 300mM NaCl FT
- 300mM NaCl SUP
- 300mM NaCl FT
- 300mM NaCl SUP
- 300mM NaCl FT
- 700mM NaCl SUP
- 700mM NaCl FT
- 700mM NaCl SUP
- 700mM NaCl FT
- 700mM NaCl SUP
- 700mM NaCl FT
- Space
- 300mM NaCl Native Elution
- 300mM NaCl LDS Elution
- 300mM NaCl Native Elution
- 300mM NaCl LDS Elution
- 300mM NaCl Native Elution
- 300mM NaCl LDS Elution
- 700mM NaCl Native Elution
- 700mM NaCl LDS Elution
- 700mM NaCl Native Elution
- 700mM NaCl LDS Elution
- 700mM NaCl Native Elution
- 700mM NaCl LDS Elution
Wet Transfer, 70V for 90 minutes at 4C
Primary antibody (anti-RRP6) 1:1000 overnight
For Wes, run duplicate lanes probing half with anti-RRP6 and half with anti-FLAG
Mass Spec samples were given to Dennis to perform S-Trap procedure; see his wiki for details.
WB with anti-RRP6 Ab
We tried to striped this membrane. But it wasn't striped enought so signal from rrp6 was still there.
Then we made a new blot with new anti-MTR4 antibody (That Domanski recommended) Ab70552
Later Nastya compared two different anti-MTR4 antibodies. For Ab70551 product concentration is 0,2 mg/mL, for Ab70552 - 1 mg/mL. 15 ml - volume of diluted Ab.
dilutions:
|| Ab70551 || Ab70552 || || 3,75 ul of stock || 0,75 ul of stock ||
So it was 1/4000 for Ab70551 but because Ab70552 is more concentrated Nastya diluted it so it's not fair comparison.
