Checking new MT302 powder
Date: 08/27/21
Cell lines:
MT302 Puro selected (4/9/2013)
MT302 Dox 24-hour induction (8/26/21)
Extraction: 20mM HEPES, pH7.4, 1% Tritonx-100, 500mM NaCl (with protease inhibitors)
Scale:50mg per IP reaction with anti-FLAG or anti-ORF1 beads
Weigh out 100mg of MT302 old or new powder, add 400ul of extraction buffer to each tube (1:4 w/v), vortex to mix
Sonicate 5 x 2 sec at 2 Amp;
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Save 5ul of clarified lysate (Input, in case for Western)
Split the rest into 2 equal parts and set up 2x 50mg IP reactions with 5ul anti-FLAG or anti-ORF1 beads
IP @ 4°C for 30’
After IP, save flow-through (FT, in case for Western)
wash 3x 1ml extraction buffer
Switch beads to fresh tubes at 2nd wash step
Elute the beads with 10ul of 1.1x LDS, 70 °C for 5’ with mixing
Save 1ul aside (E, in case for Western)
Add DTT to 50mM to all samples
Heat the samples @70°C for 10’
Run 4-12% Bis-Tris gel to analyze the elution
1) Marker
2) E-MT302 (old) anti-FLAG
3) E-MT302 (new) anti-FLAG
4) E-MT302 (old) anti-ORF1
5) E-MT302 (new) anti-ORF1
6) BSA_50ng
7) BSA_150ng
Western analysis
Load the Input (2ul), FT (2ul) and elution (1ul, 10% of total) on a 15-well 4-12% Bis-Tris gel
1) Marker
2) Input-MT302 (old)
3) FT-MT302 (old)_anti-FLAG
4) FT-MT302 (old)_anti-ORF1
5) E-MT302 (old)_anti-FLAG
6) E-MT302 (old)_anti-ORF1
7) Marker
8) Input-MT302 (new)
9) FT-MT302 (new)_anti-FLAG
10) FT-MT302 (new)_anti-ORF1
11) E-MT302 (new)_anti-FLAG
12) E-MT302 (new)_anti-ORF1
13) Marker
Wet transfer 70V for 1.5h
Blocking: TBST/5% milk, RT for 2hrs
Cut the membrane in half, between 75 and 50KD, use the two for anti-ORF2 and bottom for anti-ORF1
Upper panel: Anti-ORF2 Western
Primary Ab: Rabbit anti-ORF2 antibody (Clone 5-5, 1.03mg/ml), 1:500, 4°C, overnight
Secondary Ab: ECL anti-rabbit HRP 1:10,000, RT, 1hr
Lower panel: Anti-ORF1 Western (Abmart, 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
10 sec exposure, high sensitivity, auto tone
Strip the top panel of the membrane and reprobe it with anti-FLAG antibody
Anti-FLAG Western
Primary Ab: Mouse anti-ORF2 antibody (Sigma F3165, 4mg/ml) 1:2000, RT 1.5hr Secondary Ab: ECL anti-moudrHRP 1:10,000, RT, 1hr
1 min exposure, high sensitivity, auto tone
