Neutrophils anti-ORF1 Western
Date: 5/18/21
Two samples received on 5/13/21 • SLE1352, 20x10^6 cells • HC3319BW, 15x10^6 cells
Based on pellet size, the scale of this IP will be much less than our normal 50mg. Since we will suspend the cells in extraction buffer and sonicate them, it will be difficult to handle if the sample volume is too small. We will handle these samples as if we are doing 50mg IP.
Extraction buffer: 20mM HEPES, pH7.4, 500mM NaCl 1% Triton X-100 with protease inhibitor
Add 200ul of extraction buffer to each tube
Sonicate, 2 Amp for 3 x 2 sec
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Save 10ul of clarified lysate for Bradford Assay to determine how much material was actually used for IP Used 2.5ul for Bradford assay: SLE 4.08mg/ml and HC 2.88mg/ml. The leftover clarified lysate was enough for loading 20ug of input for Western (SLE: 5ul @ 4.08mg/ml; HC: 6.9ul @ 2.88mg/ml)
Use the rest of the lysate to set up IP with 5ul of anti-ORF1 beads per reaction
IP @ 4°C for 1 hr
After IP collect the flowthrough (“FT”) from both samples
Load same value of FT as Input for Western (SLE: 5ul; HC: 6.9ul)
Wash beads 3x 1ml of extraction buffer
Transfer beads to fresh tubes during 2nd wash
Elute beads with 10ul of LDS (“E”) @ 70°C for 5’
Anti-ORF1 Western
Add LDS (1x final) and DTT (50mM final) to gel samples as needed
Heat samples @ 70°C for 10’ before loading
Run 15-well 4-12% Bis-Tris gel
1) Marker
2) SLE_Input
3) SLE_FT
4) HC_Input
5) HC_FT
6) Space
7) SLE_E
8) HC_E
9) Space
10) MT302_5% elution of 50mg IP
11) Marker
Wet transfer, 70V for 1.5hr
Block both blots with TBST/5% milk @ RT for 1hr
Primary Ab: Mouse anti-ORF1 antibody, 1:1,000 4°C overnight (2ug/ml final concentration)
3 quick rinse with TBST followed by wash with TBST, 3x 10’ @ RT
Secondary Ab: ECL anti-mouse HRP 1:10,000, RT 1hr
3 quick rinse with TBST followed by wash with TBST, 3x 10’ @ RT
Develop the blot
5 minutes exposure, super sensitivity, auto tone
