CRC tissues anti-ORF1 IP
Date: 06/17/20
159 Metastatic Rectal Carcinoma (Liver)
174 Metastatic carcinoma (Breast) We have 174 Breast CA and 174 Breast Primary
185 MMMT (Ovary)
Scale: 50mg with 5ul of anti-ORF1 beads
Extraction buffer: 20mM HEPES, pH7.4, 1% Tritonx-100, 500mM NaCl (with protease inhibitors)
IP under the following conditions (4C time course):
1) 159T_no sonication_15’
2) 185T_no sonication_5’
3) 185T_no sonication_15’
4) 185T_no sonication_30’
5) 174T_no sonication_5’
6) 174T_no sonication_15’
7) 174T_no sonication_30’
8) 174T_with sonication_30’
9) 174 primary_with sonication_30’
Weight out 50mg aliquots of each powder; add 200ul of extraction buffer and mix by vortexing
Sonicate 2 Amp, 4x 1sec, 10J (only 8 and 9)
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Combine un-sonicated 174 and 185 lysate separately and split each into 3 aliquots (50mg each)
IP @ 4°C for 5’, 15’ and 30’ (see conditions described above)
At 5’, 15’ 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 15-well 4-12% Bis-Tris gels, load 50% of elution of gel for Western
1) 159T_no sonication_15’
2) 185T_no sonication_5’
3) 185T_no sonication_15’
4) 185T_no sonication_30’
5) 174T_no sonication_5’
6) 174T_no sonication_15’
7) 174T_no sonication_30’
8) 174T_with sonication_30’
9) 174 primary_with sonication_30’
10) MT302 _ no sonication_30’ (10% of 50mg IP; old samples saved at -20C)
11) Prestained marker_5ul
Wet transfer 70V for 1.5h
Blocking: TBST/5% milk, RT for 2hrs
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: Sonication is not necessary, IP for 15’ may be the best for capture protein