CRC tissues anti-ORF1 IP
Date: 06/10/20
References (Old experiments):
162T and 162N_anti-ORF1 IP_052819
162T and 162N_anti-ORF1 IP with mIgG control_072919
Human tissues: 100mg each Samples for IP (no sonication except the two mentioned below)
1) 162T (Liver Met B) (positive control)
2) 185T (Ovary mmmt)
3) 185T (Ovary mmmt) with sonication
4) 197T (Colon CA)
5) 197N
6) 198T (Colon tumor)
7) 198N
8) 268T
9) 268N (Normal spleen)
10) 284 (Ovary tumor)
11) 284 (Ovary tumor) with sonication
12) MT302 (positive control, load 10% of 50mg IP)
Extraction buffer: 20mM HEPES, pH7.4, 1% Tritonx-100, 500mM NaCl (with protease inhibitors)
Scale: 50mg with 5ul of anti-ORF1 beads (100mg lysate, split in halves for 5’ and 30’ time points)
Weight out 100mg aliquots of each powder; add 400ul of extraction buffer and mix by vortexing
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Also based on previous experiment: “05/28/19 - 162T and 162N anti-ORF1 IP_Sonication_Beads titration_Time course”, it seems like that sonication is not critical for extraction and 5’ IP may be sufficient, I am going to try the following conditions: IP @ 4°C for 5’ and 30’ (most samples without sonication; two samples with or without sonication)
Since we will not be able to detect Orf1p in cell lysate (Ref. 05/28/19 - 162T and 162N anti-ORF1 IP_Sonication_Beads titration_Time course), no need to save input and FT
After IP for 5’, vortex the tube, take 50% of IP reaction (IP-30) and put the rest back to the cold room for another 25’ (IP-30)
At 5’ and 30’ time points, collect the beads
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 26-well 4-12% Bis-Tris gels, load 50% of elution of gel for Western
1) Prestained marker_5ul
2) 162T-B_IP-5
3) 162T-B_IP-30
4) 185T_IP-5
5) 185T_IP-30
6) 185T+sonication_IP-5
7) 185T+sonication_IP-30
8) 197T_IP-5
9) 197T_IP-30
10) 197N_IP-5
11) 197N_IP-30
12) 198T_IP-5
13) 198T_IP-30 (IP reaction recovered at 30’ is 15% less than half)
14) 198N_IP-5
15) 198N_IP-30
16) 268T_IP-5
17) 268T_IP-30
18) 268N_IP-5
19) 268N_IP-30
20) 284T_IP-30
21) 284T_IP-5
22) 284T+sonication_IP-30
23) 284T+sonication_IP-5
24) MT302 _IP-5 (10% of 50mg IP)
25) MT302 _IP-30 (10% of 50mg IP)
26) Prestained marker_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
10 second exposure, high sensitivity, auto tone
Conclusion: Orf1p yield from 30’ IP seems to be higher than that from 5’ IP