Anti-ORF1 IP with N2102Ep and NTERA2
Date: 07/28/20
Cell lines: N2102Ep_120419 and NTERA_121019
Extraction: 20mM HEPES, pH7.4, 1% Tritonx-100, 300mM NaCl (with protease inhibitors)
Scale and powder to beads ratio: 50mg per IP reaction with 10ul of anti-ORF1(monoclonal antibody) or LaORF1 (nanobody) beads
XL nanobody to Epoxy beads after conjugation
Take aliquots of beads: 4x 10ul of anti-LaORF1-5 (08/01/19 and 10/24/19 mixed) 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 beads in 3x 1ml extraction buffer before IP
For anti-ORF1 beads, wash beads in 3x 1ml extraction buffer before IP
Anti-ORF1 IP
Weigh out 2x 100mg of each cell powder, add 400ul of extraction buffer to each tube (1:4 w/v), vortex to mix
New sonicator: Sonicate 5 x 2 sec at 2 Amp;
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Save 20ul of the input from each 100mg extract for Western (to test 2nd antibodies)
Split supernatant of each 100mg powder evenly into 2 aliquots (50mg each, one for anti-ORF1 IP and the other one for LaORF1 IP)
Set up 8x 50mg IP reactions with 10ul anti-ORF1 or LaORF1 beads
1) N2102Ep_anti-ORF1_Hua
2) N2102Ep_anti-ORF1_Mehrnoosh
3) N2102Ep_LaORF1_Hua
4) N2102Ep_LaORF1_Mehrnoosh
5) NTERA2_anti-ORF1_Hua
6) NTERA2_anti-ORF1_Mehrnoosh
7) NTERA2_LaORF1_Hua
8) NTERA2_LaORF1_Mehrnoosh
IP @ 4°C for 30’
After IP, wash 3x 1ml extraction buffer
Switch beads to fresh tubes at 2nd wash step
Elute with 10ul LDS @70 °C for 10’ with mixing
Add DTT to 50mM to LDS elution and heat @70 °C for 10’ (use 90% of elution for Sypro gel and 10% for Western)
Run 4-12% Bis-Tris gel to analyze the elution before and after precipitation (1a-4a), Sypro stain
1) Unstained Marker_1ul
2) N2102Ep_anti-ORF1_Hua
3) N2102Ep_anti-ORF1_Mehrnoosh
4) N2102Ep_LaORF1_Hua
5) N2102Ep_LaORF1_Mehrnoosh
6) NTERA2_anti-ORF1_Hua
7) NTERA2_anti-ORF1_Mehrnoosh
8) NTERA2_LaORF1_Hua
9) NTERA2_LaORF1_Mehrnoosh
10) BSA_25ng
11) BSA_100ng
12) Prestained Marker_5ul
Run 10% of the elution on 4-12% Bis-Tris gel for Western analysis
1) Prestained Marker_5ul
2) N2102Ep_anti-ORF1_Hua
3) N2102Ep_anti-ORF1_Mehrnoosh
4) N2102Ep_LaORF1_Hua
5) N2102Ep_LaORF1_Mehrnoosh
6) NTERA2_anti-ORF1_Hua
7) NTERA2_anti-ORF1_Mehrnoosh
8) NTERA2_LaORF1_Hua
9) NTERA2_LaORF1_Mehrnoosh
10) Space
11) Prestained Marker_5ul
12) N2102Ep input (25ug)
13) NTERA2 input (25ug)
14) Prestained Marker_5ul (not shown)
Wet transfer, 70V for 1.5 hours
Blot with TBST/5% milk at RT for 1-2 hrs
Anti-ORF1 Western (Abmart anti-ORF1 antibody 4H1)
Primary Ab: Mouse anti-ORF1 antibody, 1:5,000
Secondary Ab: ECL anti-mouse HRP 1:10,000
10 seconds exposure, high sensitivity, auto tone
2 minutes exposure, high sensitivity, auto tone
