In addition to the previous entry:
1) 3x pLD401 light | 100mg + 2x 200mg
2) 3x N2102Ep | 100mg + 2x 200mg
3) 3x HeLa S3 Flp-In | 100mg + 2x 200mg100 mg samples were used to prep whole cell lysis, and were transferred as such to Ydwine.
200mg samples were in duplex; 1 for gel plug and one for band excision at ~150kDa.
20ul of beads per sample of 200 mg ! Samples should be prepared with MS-grade reagents
Extraction buffer: 20mM HEPES, pH7.4, 1% TritonX-100, 500mM NaCl, 1x protease inhibitor
1. Weigh out cell powder, equilibrate at RT for 30 secs, add extraction buffer to each tube (1:4 w/v), vortex to mix
2. Sonicate 5 x 2 sec at 4 (!!!) Amp – this setting at ERIBA ~equals to 2 Amp at RU.
3. Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R). Move cleared lysate to a clean 1.5ml tube. Take 10ul input for WB
4. In mean time, wash beads with 3x 1mL cold extraction buffer
7. Set up IP reaction(s) with 5ul of antibody conjugated Dynabeads
8. IP @ 4°C for 30’
In mean time, measure protein concentration of lysates, dilute to one concentration, add lds to 1x final concentration and save on ice.
9. Save the flow through for WB
10. Wash 3x 1ml cold extraction buffer
11. Switch beads to fresh tubes at 2nd wash step
12. Elute the beads with 10ul 1.1x LDS, 70 °C for 5’ with mixing in low binding protein tubes
13. Quick spin, transfer elution to fresh tubes low binding
14. Add DTT to 50mM to all samples and heat @70 °C for 10’
Samples were transferred to Ydwine hereafter.