197T and 197N anti-ORF1 IP
Date: 06/24/21
Samples for IP: 197T (Colon CA) and matching normal 197N
Extraction buffer: 20mM HEPES, pH7.4, 1% Tritonx-100, 500mM NaCl (with protease inhibitors)
Scale: 50mg with 5 or 10ul of anti-ORF1 beads or mIgG control
Weight out 2x100mg 197T and 100mg 197N; add 400ul of extraction buffer and mix by vortexing
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Sonicate @ 2Amp, 5x1 sec
Spin @ 20k rcf , 4°C for 10’
Collect the clarified lysate and set up 50mg IPs
1) 197T_anti-ORF1_5ul beads
2) 197T_anti-ORF1_10ul beads
3) 197T_mIgG_5ul beads
4) 197T_mIgG _10ul beads
5) 197N_anti-ORF1_5ul beads
6) 197N_anti-ORF1_10ul beads
IP @ 4°C for 30’
Wash 3x 1ml extraction buffer
Transfer beads to fresh tubes at 2nd wash step
Elute with 10ul of LDS @ 70°C for 5’
Add DTT to 50mM to all samples and heat @ 70°C for 10’ Run elution on 15-well 4-12% Bis-Tris gels, load 20% of elution for Sypro stain and 50% of elution of gel for Western
Gel 1 for Sypro stain
1) Unstained marker_1ul
2) 197T_anti-ORF1_5ul beads
3) 197T_anti-ORF1_10ul beads
4) 197T_mIgG_5ul beads
5) 197T_mIgG _10ul beads
6) 197N_anti-ORF1_5ul beads
7) 197N_anti-ORF1_10ul beads
8) BSA_10ng
9) BSA_50ng
10) Prestained marker_5ul
Gel 2 for anti-ORF1 Western
1). Prestained marker_5ul
2) 197T_anti-ORF1_5ul beads
3) 197T_anti-ORF1_10ul beads
4) 197T_mIgG_5ul beads
5) 197T_mIgG _10ul beads
6) 197N_anti-ORF1_5ul beads
7) 197N_anti-ORF1_10ul beads
8) Prestained marker_2.5ul
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
30 seconds exposure, high sensitivity, auto tone
The yields of ORF1p were similar using either 5ul (lane 2) and 10ul (Lane 3) for 50mg IP
Similar result was observed for 162 and 174
Wiki entry 05/28/19 - 162T and 162N anti-ORF1 IP_Sonication_Beads titration_Time course
See Western of elution at the end of the file, first Western image (using 10, 20 and 25ul beads for 100mg IP)
Wiki entry May 5, 2021 - Effects of ZnCl2 on IP specificity using CRC tissue
See Western of elution at the end of the file (using 5, and10ul beads for 50mg IP)