Cell line: HEK-293 EXOSC10-3xFLAG (RRP6-3xFLAG)
Before beginning:
1. place sonicator probe in cold room
2. mix extraction buffer aliquots with protease inhibitors; place remaining buffer on ice in preparation to wash beads
Extraction/Wash Buffers:
| Name of buffer | Temp | Components | Volume per sample | Total volume |
|---|---|---|---|---|
| Wash buffer | ice | 20mM HEPES, pH 7.4, 100mM MgCl2, 1% Triton X-100 (v/v) | 4 ml before IP 3 ml after IP | 21 ml |
| Extraction buffer | RT | Wash buffer + 4 ul of protease inhibitor (for 100 mg of powder | 400 ul | 1200 ul |
| Elution buffer (low percent of detergent) | ice | 20mM HEPES, pH 7.4, 100mM MgCl2, 0,1% Triton X-100 (v/v | 25 ul of buffer + 5 ul of 5 mg/ml 3xFLAG peptide. For each sample 10 ul | 30 ul |
| No detergent buffer | RT | 20mM HEPES, pH 7.4, 100mM MgCl2 | 20 ul for each | 60 ul |
Scale: "total 300 mg in 30 ul" of anti-3xFLAG beads "because of triplication" (100 mg with 10 ul per sample)
Washing beads
Optional: wash the beads before weighing powder (or during spin). Wash them separately. Add 50 ul of wash buffer to each tube first and then add beads (just tip to make it easier). 3x 10 ul of beads; each in a 2 ml safe-lock Eppendorf tube, 4x 1 ml wash in appropriate wash buffer (wash buffers do not have protease inhibitor added and should be on ice). After all washes place tubes with beads on ice
Powder part
Label 3x 2ml safe-lock Eppendorf tubes with numbers (it is necessary to label tubes before cooling), then open and pre-cool them in LN2; also pre-cool weighing tools and large tweezers.
Take powder from -80C and place in liquid nitrogen
Weigh out 3x 100mg RRP6-3x FLAG in 2ml safe-lock eppendorf tubes, do not hold for long time powder in RT (avoid melting).
Do not forget to add protease inhibitor. Add 400 ul of appropriate extraction buffer (1:4, w/v 100 mg in 400 ul) 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
Sonication
Sonicate in cold room@ 4 Amp, 5x 2 sec; Then repeat 1x (this is the sonication regime we determined was necessary for 700 mM NaCl samples). Write down the information from sonication.
Spin @ full speed in benchtop centrifuge (~21k rcf) @ 4°C for 10’ (Eppendorf Centrifuge 5417R);
Immunoprecipitation (Save 20 ul supernatant of each samples for Western analysis if needed(“SUP” or “input”); place on ice or at -20C
Use remaining clarified lysate to set up 3x 100mg IP reactions – lysate is added to the tubes of beads and not the other way around Incubate with 10 ul pre-washed beads (scale = 10ul beads per 100mg powder
IP @ 4°C for 1h with rotation (cold room); During IP 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 (you can save 20ul of FT for Western if needed). Discard remaining FT Wash beads with 3x 1ml wash 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
Make a 1mg/ml 3xFLAG peptide. For 3 samples you need 30 ul of 1mg/ml 3xFLAG peptide in elution buffer (low detergent buffer), the stock of 3xFLAG peptide is 5 mg/ml. Dilute 5 ul of stock in 25 ul of buffer. Add 10ul 1mg/ml 3xFLAG peptide in elution buffer to each tube.
Elute for 15 min at RT with mixing in Eppendorf Thermomixer
Place on a magnet; collect the supernatant (native eluate fraction) in a fresh tube.
Add 20 ul of buffer without detergent per tube to wash the beads; collect this fraction and add to the native eluate fraction. It will be 30 ul (10 + 20) of each native elution sample. Use 6 ul of each native elution and load them together (in one well) on protein gel for Blue-Silver analysis. The remaining individual samples are sent for MS.
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.
Sample preparation
Add 4x LDS and 1M DTT (final DTT concentrations 50mM) to gel samples
| native elution | total 24 ul |
| 6 ul | LDS 4x |
| 15 ul | sample |
| 1,2 | DTT |
| 1,8 ul | ddH20 |
| LDS elution | total 24 ul |
| 19,5 ul | sample |
| 1,2 | DTT |
| 3,3 ul | LDS 1.1x |
| mock native elution | total 24 ul |
| 6 ul | LDS 4x |
| 15 ul | sample |
| 1,2 | DTT |
| 1,8 ul | ddH20 |
| mock LDS elution | total 24 ul |
| 20 ul | sample |
| 1,2 ul | DTT |
| 2,8 ul | LDS 1.1x |
Heat samples @ 70°C for 10’ to denature
Run samples on 15-well 4-12% Bis-Tris gel for Blue-Silver stain.
Combined Native elution MgCl2 = 5 ul of native elution from each replicate pooled. Total 15 ul
LDS combined elution = 6,5 ul of LDS elution from each replicate pooled. Total 19,5 ul
- Space (11 ul LDS 1.1 x)
- Marker 5 ul
- Combined Native elution MgCl2 (1-Nastya)
- LDS combined elution (1-Nastya)
- Combined Native elution MgCl2 (2-Luciano)
- LDS combined elution (2-Luciano)
- mock native elution 15 ul
- mock LDS elution 20 ul
- Space (11 ul LDS 1.1 x)
- BSA 50 ng
- BSA 200 ng
