Capturing ORF2p with anti-ORF2 beads from N2102Ep
Date: 1/22/20
Cell lines:
N2102Ep (Cell from Gerald, 50% of cells from a confluent T75 flask or cell powder prepared at RU)
HeLa (Cell from Gerald, 50% of cells from a confluent T75 flask): negative control
MT302: positive control
Scale:
3000ug cell extract of 2102Ep from Gerald with 20ul of anti-ORF2 beads*
- Based on our previous result, 3000ug of total protein is similar to the scale of 100mg IP.
100mg IP of N2102Ep powder (record the amount of total protein per IP) with 20ul of anti-ORF2 beads
Extraction buffers: with protease inhibitors 1:100; RNasin 1:250 in extraction buffer and 1:1000 in wash buffer (RNasin was added to both extraction and wash buffer in Maria’s experiment).
• L1 buffer: 20mM HEPES, pH7.4, 1% Tritonx-100, 500mM NaCl
• NP40 buffer: 25mM Tris-HCl pH 7.5, 150mM NaCl, 5mM MgCl2, 1mM DTT, 5 % glycerol, 1 % Nonidet® P-40
Resuspend each N2102Ep and HeLa pellet from Gerald in 400ul of NP40 buffer
100mg of N2102Ep powder in 400ul of L1 or NP40 extraction buffer; 50mg of MT302 powder in 200ul of L1 buffer
Sonicate @ 2 Amp for 5x 2sec
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Collect the clarified lysate
Save 10ul for Western and Bradford assay (to determine protein concentration)
Use the rest to set up anti-ORF2 IP (20ul per IP except MT302)
1) 2102Ep_Gerald_NP40
2) N2102Ep_RU_NP40
3) N2102Ep_RU_L1
4) HeLa_Gerald_NP40
5) MT302_L1 (50mg IP with 5ul of beads)
IP @ 4°C for 1h
Save 10ul of the flow-through (“FT”)
After IP, wash 3x 1ml extraction buffer
Switch beads to fresh tubes at 2nd wash step
Elute beads with 10ul of 1.1x LDS @70°C for 5’
Collect the elution for Western
To all Western samples, add DTT to 50mM and heat @70°C for 10’
Run 15-well 4-12% Bis-Tris gel for Western
1) Marker
2) Input_2102Ep_Gerald_NP40_25ug
3) FT_2102Ep_ Gerald_NP40_25ug
4) Input_N2102Ep_RU_NP40_25ug
5) FT_N2102Ep_RU_NP40_25ug
6) Input_N2102Ep_RU_NP40_25ug
7) FT_N2102Ep_RU_NP40_25ug
Skip Input and FT of HeLa in order to fit all samples in 15-well gel
8) Input_MT302_2.5ug
9) FT_MT302_2.5ug
10) Marker
11) E_2102Ep-Gerald_NP40
12) E_N2102Ep_RU_NP40
13) E_N2102Ep_RU_L1
14) E_HeLa-Gerald_NP40
15) E_MT302_L1 (10% of Elution)
Wet transfer, 70V for 2h
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, RT, 5hr
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
30-second exposure, high sensitivity, auto tone
1-minute exposure, super sensitivity, auto tone
3-minute exposure, super sensitivity
