LaCava Research Wiki

June 2021

the bead was prepared by me and Leila, procedure is here: https://macromolecule-child.rockefeller.edu/#26%20May%2021_N2102EP%20Drug%20Assay%20Prep_Dynabeads%20Testing

Place sonicator arm in the cold room before beginning;

Extraction/Wash Buffer: L1 buffer

bead

1. Hua's bead
2. Old eriba bead
3. new eriba bead

Procedure:

1. Weigh out 3x 50mg of N2102ep cell powder
2. Add 400ul of extraction buffer with 1x protease inhibitor to each tube
3. vortex to mix
4. Sonicate 5 x 2 sec at 4 Amp
5. Spin @ 20k rcf, 4°C for 10’;
6. wash beads during spin, 3x 1ml appropriate wash buffer, Set up IP reactions with 10ul of beads each
7. IP @ 4°C for 30’
8. After IP, wash with 3x 1ml extraction buffer
9. Switch beads to fresh tubes at 2nd wash step
10. Elute the beads with 10ul of 1.1x LDS, 70 °C for 5’ with mixing;
11. Collect the eluation
12. Add DTT to final concentration of 50mM heat @70 °C for 10’.
13. Run 12-well 4-12% Bis-Tris gel to analyze the elution
    • Lane 1: Blank
    • Lane 2: Blank
    • Lane 3:ladder
    • Lane 4: Abmix Input
    • Lane 5:Abmix FT
    • Lane 6: Hua's beads
    • Lane 7: old ERIBA beads
    • Lane 8: new ERIBA beads
    • Lane 9: Blank

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