Anti-ORF1 Western of elution_Input_FT in N2102Ep, NTERA and PA-1 PC and DC
Date: 06/16/20 - 06/25/20
Samples are from anti-ORF1 IP that Lars and Hua did in the middle of March 2020 June 9, 2020 - Summary of anti-ORF1p RNA extractions by Lars
Protein (in box -20) This box was found in -80 freezer as well
o Input (10 µl = 2.5%)
o FT (take 10ul and save the rest)
o Elution in LDS, no DTT (10% of beads)
Western on I, FT, E as usual
Sample #: 1. N2102Ep 2. N2102Ep 3. N2102Ep 4. N2102Ep 5. NTERA2 6. NTERA2 7. NTERA2 8. NTERA2 9. PA-1 PC 10. PA-1 PC 11. PA-1 PC 12. PA-1 PC 13. PA-1 DC 14. PA-1 DC 15. PA-1 DC 16. PA-1 DC
Blot 1: all elutions (16 samples) + positive control
Blot 2: Input and FT of N2102Ep and NTERA2 (16 samples) + positive control
Blot 3: Input and FT of PA-1 PC and DC (16 samples) + positive control
Western blot of elution (Blot 1: all elutions (16 samples) + positive control)
Use all 10ul of elution, add 1ul of 0.5M DTT, Heat @ 70C for 10’, run 26-well Bris-Tris gel
Lane 1: Marker
Lane 2-17: E1-16
Lane 18: Marker
Lane 19: 2.5% of MT302 elution (50mg anti-ORF1 IP)
Lane 20: 10% of MT302 elution (50mg anti-ORF1 IP)
Lane 21: Marker
Wet transfer 70V, 2hr
Cut the membrane in half, between 75 and 50KD, use the top for anti-ORF2 and bottom for anti-ORF1
Block the membranes in TBST/5% milk @ RT, 1hr
Upper panel: Rabbit anti-ORF2 Western (Clone 5)
Primary Ab: Rabbit anti-ORF2 antibody (Clone 5, 0.325mg/ml), 1:500, 4°C, overnight
Secondary Ab: ECL anti-rabbit HRP 1:10,000, RT, 1hr
Lower panel: Anti-ORF1 Western (Abmart, 4H1, 2mg/ml) Primary Ab: Mouse anti-ORF1 antibody, 1:5,000, 4°C, overnight
Secondary Ab: ECL anti-mouse HRP 1:10,000, RT 1hr
Anti-ORF1, standard sensitivity, 10 second exposure
/Users/hjiang01/Box/Wiki_raw_data/Hua's raw data/N2102Ep_NTERA_PA1_anti-ORF1 Western_E_061720/20200617_anti-ORF1_std_10s_auto.png
Anti-ORF2, super sensitivity, 3 min exposure
/Users/hjiang01/Box/Wiki_raw_data/Hua's raw data/N2102Ep_NTERA_PA1_anti-ORF1 Western_E_061720/20200617_anti-ORF1_super_3m.png
Western blot of input and FT
Blot 2, 3: Use 25ug of total protein of input and FT, add 50mM DTT (final concentration), Heat @ 70C for 10’, run 26-well Bris-Tris gel. There was an extra marker on blots 3 to distinguish the two blots.
Blot 2 Samples 1-8 (N2102Ep and NTERA, Input and FT next to each other, 4 replicates each)
Two empty lanes (not shown)
Lane 1: Marker
Lane 2-17: Input/FT 9-16 (25ug each)
Lane 18: Marker
Lane 19: 2.5ug of MT302 lysate
Lane 20: 10ug of MT302 lysate
Lane 21: 25ug of MT302 lysate
Marker (not shown)
Blot 3 Samples 9-16 (PA-1 PC and DC, Input and FT next to each other, 4 replicates each)
Marker (not shown)
Space (not shown)
Lane 1: Marker
Lane 2-17: Input/FT 9-16 (25ug each)
Lane 18: Marker
Lane 19: 2.5ug of MT302 lysate
Lane 20: 10ug of MT302 lysate
Lane 21: 25ug of MT302 lysate Marker (not shown)
Wet transfer 70V, 2hr
Block the membranes in TBST/5% milk @ RT, 1hr
Anti-ORF1 Western (Abmart, 4H1, 2mg/ml)
Primary Ab: Mouse anti-ORF1 antibody, 1:5,000, 4°C, overnight
Secondary Ab: ECL anti-mouse HRP 1:10,000, RT 1hr
Blot 2 N2102Ep (lane 2-9) and NTERA2 (lanes 10-17); high sensitivity, 10 second exposure
/Users/hjiang01/Box/Wiki_raw_data/Hua's raw data/N2102Ep_NTERA_PA1_anti-ORF1 Input_FT_061920/20200619_1-8 high_10s_auto.png
Blot 3 PA-1 PC (lane 2-9) and PA-1 DC (lanes 10-17); high sensitivity, 10 second exposure
/Users/hjiang01/Box/Wiki_raw_data/Hua's raw data/N2102Ep_NTERA_PA1_anti-ORF1 Input_FT_061920/20200619_9-16 high_10s_auto.png
Strip the blots 2 and 3, reprobe with higher primary and secondary antibody titers Block the membranes in TBST/5% milk @ RT, 2hr
Anti-ORF1 Western (Abmart, 4H1, 2mg/ml) Primary Ab: Mouse anti-ORF1 antibody, 1:1,000, 4°C, overnight Secondary Ab: ECL anti-mouse HRP 1:5,000, RT 1hr
When I developed the blots, the background on both blots were dirty (not shown). Add 20 minutes wash in TBST, the bacdground was gone, but ORF1p signal was weak. Did the following and redevelped the blots.
Wash 1x in TBST for 10’
Incubate with secondary Ab: ECL anti-mouse HRP 1:3,000, RT for 30’
3 quick rinse in TBST
Wash 3x TBST for 10’
Add ECL substrate and develop the membrane again
Blot 2 N2102Ep (lane 2-9) and NTERA2 (lanes 10-17); high sensitivity, 1 minute exposure
/Users/hjiang01/Box/Wiki_raw_data/Hua's raw data/N2102Ep_NTERA_PA1_anti-ORF1_1;1000_Input_FT_062420/1-8 2nd set_anti-mouse 3000_more wash/20200625_1-8-2nd_3000_super_5m_auto.png
Blot 2 N2102Ep (lane 2-9) and NTERA2 (lanes 10-17); super sensitivity, 5 minutes exposure
/Users/hjiang01/Box/Wiki_raw_data/Hua's raw data/N2102Ep_NTERA_PA1_anti-ORF1_1;1000_Input_FT_062420/1-8 2nd set_anti-mouse 3000_more wash/20200625_1-8-2nd_3000_super_5m_auto.png
Blot 3 PA-1 PC (lane 2-9) and PA-1 DC (lanes 10-17); super sensitivity, 5 minutes exposure
/Users/hjiang01/Box/Wiki_raw_data/Hua's raw data/N2102Ep_NTERA_PA1_anti-ORF1_1;1000_Input_FT_062420/9-16 2nd set_anti-mouse 3000_more wash/20200625_9-16 bottom_super_1m_1_1.tif