Anti-ORF1 PO testing anti-LaORF1 beads with XL
Date: 06/29/20
Cell lines: MT302 Light _05/24/14, N2102Ep_12/10/19 and NTERA2_12/06/19
Extraction/wash buffer: 20mM HEPES, pH7.4, 1% Tritonx-100, 500mM NaCl (with protease inhibitors)
Scale: 25 or 50 mg per IP reaction with 10ul of anti-LaORF1 beads per 50mg
Beads:
1) anti-LaORF1-5 (nanobody beads, 08/01/19, XL nanobody to beads)
2) anti-LaORF1-5 (nanobody beads, 10/24/19, XL nanobody to beads)
3) anti-LaG94-10 (nanobody beads, 10/24/19, XL nanobody to beads)
XL nanobody to Epoxy beads after conjugation
Take aliquots of beads: 5ul of anti-LaORF1-5 (08/01/19), 30ul of anti-LaORF1-5 (10/24/19), and 5ul of anti-LaG94-10 (nanobody against GFP, 10/24/19) beads, wash once in 1ml of 1xPBS
Resuspend beads in 20mM DMP in Borate buffer (2ml per reaction with 100ul of beads; make this right before use when beads are washing; I am using more DMP solution than needed to ensure that the beads will be mixing well during the XL step below)
Perform XL reaction @ RT on the wheel for 30’.
After 30’, remove the XL solution
Wash beads with 1ml of PBST
Wash beads again with 1ml of TBST for 5’ @ RT
Wash beads again with 1ml of PBST
Wash beads with 1ml of 1x PBS
Wash all beads in 3x 1ml extraction buffer before IP
Anti-ORF1 IP
Weigh out 50mg MT302, 100mg of N2102Ep and 50mg of NTERA2, add 400ul of extraction buffer with protease inhibitors per 100mg of powder (1:4 w/v), vortex to mix
Sonicate 5 x 2 sec at 2 Amp;
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Collect the supernatant, split it evenly into 6 aliquots
Set up 25mg or 50mg IP reactions with 10ul of beads per 50mg reaction
1) MT302_25mg_5ul LaORF1_080119
2) MT302_25mg_5ul LaORF1_102419
3) N2102Ep_25mg_5ul LaORF1_102419
4) N2102Ep_25mg_5ul LaG_102419 (negative control)
5) N2102Ep_50mg_10ul LaORF1_102419
6) NTERA2_50mg_10ul LaORF1_102419
IP @ 4°C for 30’
After IP, wash 3x 1ml extraction buffer
Switch beads to fresh tubes at 2nd wash step
Elute the beads with 10ul of LDS, 70 °C for 5’ with mixing
Collect the eluate
Add DTT to 50mM to all samples and heat @70 °C for 10’
Run 4-12% Bis-Tris gel to analyze the elution, Sypro stain
1) Unstained Marker_1ul
2) MT302_25mg_5ul LaORF1_080119
3) MT302_25mg_5ul LaORF1_102419
4) N2102Ep_25mg_5ul LaORF1_102419
5) N2102Ep_25mg_5ul LaG_102419 (negative control)
6) N2102Ep_50mg_10ul LaORF1_102419
7) NTERA2_50mg_10ul LaORF1_102419
8) BSA_25ng
9) BSA_100ng
10) Space
11) Prestained Marker_5ul
2 second exposure
Conclusion: Both batches of LaORF1 worked for anti-ORF1 IP. The 2nd batch (10/24/20) seems to have a bigger problem with nanobody leakage even after DMP XL.