Anti-FLAG IP with MT646, MT647, LD564, MT302 and LD401
Date: 09/29/21
Constructs to test:
pMT646: S221_08/09/21, low_08/24/21 and high_08/25/21(Marty/Kaay)
pMT647_08/25/21(Marty/Kaay)
pLD564_08/25/21(Marty/Kaay)
MT302_08/26/21 (Hua/Leila)
MT302 Light_05/02/14 (Marty)
LD401 Light_05/02/14 (Marty)
Extraction: 20mM HEPES, pH7.4, 1% Tritonx-100, 500mM NaCl (with protease inhibitors)
Scale: 50mg per IP reaction with 5ul anti-FLAG
Weigh out 50mg of each 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)
Collect the clarified lysate
Save 10ul of clarified lysate (Input for Western)
Use the rest to set up 2x 50mg IP reactions with 5ul anti-FLAG
IP @ 4°C for 30’
After IP, save flow-through (FT 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 for Western)
Add DTT to 50mM to all samples
Heat the samples @70°C for 10’
Run 15-well 4-12% Bis-Tris gel to analyze the elution
1) Marker
2) E_MT646_S221
3) E_MT646_low
4) E_MT646_high
5) E_MT647
6) E_MT564
7) E_MT302
8) E_MT302 Light
9) E_LD401 Light
10) BSA_50ng
11) BSA_150ng
Run 26-well 4-12% Bis-Tris gel to analyze the Input (2ul), FT (2ul) and elution (10%)
1) Marker
2) Input_MT646_S221
3) FT_MT646_S221
4) Input_MT646_Low
5) FT_MT646_Low
6) Input_MT646_high
7) FT_MT646_high
8) Input_MT647
9) FT_MT647
10) Input_MT564
11) FT_MT564
12) Input_MT302
13) FT_MT302
14) Input_MT302 Light
15) FT_MT302 Light
16) Input_LD401 Light
17) FT_LD401 Light
18) E_MT646_S221
19) E_MT646_Low
20) E_MT646_high
21) E_MT647
22) E_MT564
23) E_MT302
24) E_MT302 Light
25) E_LD401 Light
26) Marker
Run the gel longer till 15kd marker to the bottom of the gel
Wet transfer 70V for 1.5h
Block in TBST/5% milk @ RT for 2h
Cut the membrane into 4 pieces, below 100KD, above 50KD and below 37KD marker
Anti-FLAG Western (~150KD)
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-Lamin B1 Western (~68KD)
Primary Ab: rabbit anti-Lamin B1 (Cell Signaling #13435), 1:1,000, 4°C overnight
Secondary Ab: ECL anti-rabbit HRP 1:10,000, RT for 1hr
Anti-ORF1 Western (~40KD)
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
Anti-PCNA Western (~36KD)
Primary Ab: mouse anti-PCNA antibody (Santa Cruz, sc-56) 1:1,000 (old Ab that has been sitting in the fridge for years)
Secondary Ab: ECL anti-mouse HRP 1:10,000
20 seconds exposure, high sensitivity, auto tone
4 minutes exposure, high sensitivity, show Lamin B1
