pMT646 and pMT647 anti-FLAG IP
Date: 06/16/21
Cell lines:
pMT646, ORFeus ORF2p-3xFlag (clean tags) CMV promoter in pCEP4 Puro
pMT647, ORF2p only ORFeus ORF2p-3xFlag (clean tags) CMV promoter in pCEP4 Puro
Scale: 400mg per experiment (with 20ul of anti-FLAG beads) Extraction buffer: 20mM HEPES, pH7.4, 1% Tritonx-100, 500mM NaCl + protease inhibitors
Two 15ml Falcon tubes from Marty (1x pMT646 and 1x pMT647, ~400mg each), add 1600ul of extraction buffer to each (1:4 w/v), vortex to mix
Sonicate 5x 2 sec @ 2 Amp, repeat once
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Combine the clarified lysate from the same cell line in one tube
Save 10ul lysate (“Input”) for Western (Determine protein concentration by Bradford assay and load 25ug for Western)
Add 20ul of anti-FLAG beads
Incubate @ 4°C for 30’
After 30’, save 4ul supernatant (“Sup”) for Western (load 2ul)
Wash 3x 1ml extraction buffer (transfer the beads to fresh tubes during 2nd wash)
Native elution:
a) Elute with 20ul of 1mg/ml 3x FLAG peptide, RT for 15’ with mixing
b) Wash beads with 20ul of extraction buffer
c) Combine the elution and wash (40ul total for 400mg IP)
LDS wash of the beads:
a) Add 40ul of 1.1x LDS to beads, incubate @ RT for 10’ with mixing
b) Collect the LDS wash (40ul for 400mg IP)
Add LDS and DTT as needed to the samples (final 1x LDS and 50mM DTT)
Heat all samples @ 70°C for 10’
Gel analysis of elution and wash
Load 10ul (25% of the elution and wash, ~100mg IP); Coomassie stain
1) Marker
2) E_MT646
3) LDS_MT646
4) E_MT647
5) LDS_MT647
6) BSA_50ng
7) BSA_150ng
Western analysis
Load 2ul of input and FT and 4ul of diluted elution (equivalent to 2% of the total elution)
15-well 4-12% Bis-Tris gel
1) Marker_5ul
2) Input_MT646
3) FT_MT646
4) Input_MT647
5) FT_MT647
6) Marker_2ul (not shown)
7) E_MT646 (1ul, 10mg scale)
8) LDS_MT646 (1ul, 10mg scale)
9) E_MT647 (1ul, 10mg scale)
10) LDS_MT647 (1ul, 10mg scale)
11) Space
12) E_MT302 (old elution of MT302 anti-ORF1 IP, 10% of 100mg scale; control for ORF1p) 13) Marker_2ul (not shown)
Wet transfer 70V for 1.5h
Cut the membrane between 75KD and 50KD markers
Block in TBST/5% milk @ RT for 1hr
Anti-FLAG Western
Primary Ab: mouse anti-FLAG (Sigma F3165), 1:2,000, 4°C overnight
Secondary Ab: ECL anti-mouse HRP 1:10,000, RT for 1hr
Anti-ORF1 Western
Primary Ab: mouse anti-ORF1 from Abmart (4H1), 1:5,000, 4°C overnight
Secondary Ab: ECL anti-mouse HRP 1:10,000, RT for 1hr
10 seconds exposure, high sensitivity, auto tone
