AIM/HYPOTHESIS
To test whether we can pullout Orf2p from large scale N2102Ep anti-ORF2 IP
MATERIALS
Cell lines:
N2102Ep
MT302 Transient
Scale: 1g N2102Ep with 10ul anti-ORF2 beads per 50mg IP 50mg MT302 with 5ul of anti-ORF2 beads (50mg IP, load 10% of elution for Western) Extraction buffer: 20mM HEPES, pH7.4, 1% Tritonx-100, 500mM NaCl (with protease inhibitors 1:100). Add RNasin 1:250 in extraction buffer and 1:1000 in wash buffer.
METHODS and RESULTS
Weight out powder (described in “Scale”); add extraction buffer (1:4 w:v), vortex to mix
Sonicate @ 2 Amp for 5x 2sec - x 4 times (This is the same % that we have been using for anti-ORF2 IPs on PA-1) – Do just once for 50 mg of MT302
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R),
Combine all clarified lysate from N2102Ep
Save 10ul for Western and protein assay
Use the rest of the lysate to set up anti-ORF2 IP (Split the rest of N2102Ep lysate evenly into 4 aliquots)
1) N2102Ep (4x 250mg) 2) MT302 (1x 50mg)
For detecting ORF2 in N2102EP use double the amount of anti-ORF2 beads than for MT302 (MT302: 50mg/5ul anti-ORF2 beads; N2102Ep: 50mg/10ul anti-ORF2 beads) IP @ 4°C for 30’
Combine FT from all 4 tubes of N2102Ep
Save 10ul of the flow-through (“FT”)
After IP, wash 3x 1ml extraction buffer
Switch beads to fresh tubes at 2nd wash step; combine 2x 250mg of N2102Ep
Elute beads with 10ul of 1.1x LDS @70°C for 5’
Collect the elution for Western
MT302: 50mg IP, 10ul elution
N2102Ep: 1g IP, 20ul elution
Take 1ul out of 20ul (5% or 50mg, equivalent to MT302 scale), add 9ul of 1x LDS; This will be “E_2102Ep_diluted”; 50mg in 10ul, similar to what we did for MT302
N2102Ep_diluted: 50mg IP, 10ul final vol.
Measure the protein concentration in input and FT and load 25ug for Western (25 and/or 2.5ug from MT302)
To all Western samples, add DTT to 50mM and heat @70°C for 10’
Run 15-well 4-12% Bis-Tris gel (2 gels) for Western (loading order right before Western images)
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
Primary Ab: Rabbit anti-ORF2 antibody (Clone 9-7, 1.39mg/ml), 1:1000, 4°C, overnight
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
Western results
Gel1 (try to detect ORF2p from N2102)
1) Marker
2) Input_N2102Ep_25ug
3) FT_N2102Ep_25ug
4) Input_MT302_25ug
5) FT_MT302_25ug
6) Input_MT302_2.5ug
7) FT_MT302_2.5ug
8) Marker (not shown)
9) Space
10) E_N2102Ep_19ul (95%, 950mg)
11) Space
12) E_MT302_1ul (10%, 5mg)
13) Marker (not shown)
Anti-ORF2 Western (Clone 9-7), high sensitivity, 20s exposure, auto tone; Showing what is in N2102Ep elution lane (N2102Ep lane is overloaded, diffused to adjacent lanes)
Anti-ORF2 Western (Clone 9-7), high sensitivity, 6m exposure, show potential Orf2p in Input and FT
Anti-ORF1 Western, standard sensitivity, 10s exposure, auto tone
Gel2 (try to compare ORF2 expression level in N2102Ep vs MT302)
Reprobe the top panel of Gel 1
Strip the top panel of gel 1 and re-probe it with anti-ORF2 (Clone 11-1)
Primary Ab: Rabbit anti-ORF2 antibody (Clone 11-1, 0.445 mg/ml), 1:500, 4°C, overnight Secondary Ab: ECL anti-rabbit HRP 1:10,000, RT, 1hr
Anti-ORF2 Western (Clone 11-1), high sensitivity, 30s exposure, auto tone
Anti-ORF2 Western (Clone 11-1), super sensitivity, 5m exposure, auto tone
Strip the top panel of gel 1 again and re-probe it with anti-ORF2 (Clone 5-5)
Primary Ab: Rabbit anti-ORF2 antibody (Clone 5, 0.325 mg/ml), 1:500, 4°C, overnight
Secondary Ab: ECL anti-rabbit HRP 1:10,000, RT, 1hr
Anti-ORF2 Western (Clone 5-5), high sensitivity, 30s exposure, auto tone
Anti-ORF2 Western (Clone 5-5), super sensitivity, 5m exposure, auto tone
Gel2 (try to compare ORF2 expression level in N2102Ep vs MT302)
1) Marker
2) Input_N2102Ep_25ug
3) FT_N2102Ep_25ug
4) Input_MT302_25ug
5) FT_MT302_25ug
6) Space
7) Marker
8) E_N2102Ep_diluted_1ul (10%, 5mg)
9) E_N2102Ep_diluted_2ul (20%, 10mg)
10) E_N2102Ep_diluted_5ul (50%, 25mg)
11) Space
12) E_MT302_1ul (10%, 5mg)
13) E_MT302_2ul (20%, 10mg)
14) E_MT302_5ul (50%, 25mg)
15) Marker (not shown)
Anti-ORF2 (Clone 9-7)and anti-ORF1 IP Western, high sensitivity, 20s exposure, auto tone; compare L1 expression in N2102Ep vs MT302
Strip the top panel of gel 1 again and re-probe it with anti-ORF2 (Clone 5-5)
Primary Ab: Rabbit anti-ORF2 antibody (Clone 5, 0.325 mg/ml), 1:500, 4°C, overnight Secondary Ab: ECL anti-rabbit HRP 1:10,000, RT, 1hr
Anti-ORF2 Western (Clone 5), high sensitivity, 30s exposure, auto tone
Anti-ORF2 Western (clone 5), super sensitivity, 5m exposure, auto tone
Reprobe the top panel of Gel 2
Strip the top panel of gel 2 and re-probe it with different clone anti-ORF2 (Clone EN49)
Primary Ab: Rabbit anti-ORF2 antibody (Clone 49, 0.35 mg/ml), 1:500, 4°C, overnight Secondary Ab: ECL anti-rabbit HRP 1:10,000, RT, 1hr
Anti-ORF2 Western (Clone EN49), high sensitivity, 30s exposure, auto tone
Anti-ORF2 Western (Clone 49), super sensitivity, 5m exposure, auto tone
Strip the top panel of gel 2 again and re-probe it with different clone anti-ORF2 (Clone 11-1)
Primary Ab: Rabbit anti-ORF2 antibody (Clone 11-1, 0.445 mg/ml), 1:500, 4°C, overnight
Secondary Ab: ECL anti-rabbit HRP 1:10,000, RT, 1hr
Anti-ORF2 Western (Clone 11-1), high sensitivity, 30s exposure, auto tone
Anti-ORF2 Western (Clone 11-1), super sensitivity, 5m exposure, auto tone
Analysis N2102 IP by MS
Beads were saved after anti-ORF2 IP and were washed with LDS again and loaded on 4-12% Bis Tris gel
1) Marker
2) Space
3) Beads from 250mg of N2102Ep IP (1/4 of total beads)
4) Space
5) Beads from 50mg of MT302 IP
6) Space
7) BSA_50ng
8) BSA_150ng
Wash the rest of the beads from N2102Ep IP, treated for MS, ran the sample on 4-12% Bis Tris gel and cut 4 regions for MS
1) top, light stained doublet (a)
2) “empty" middle area (b)
3) lower, heavy stained doublet (c)
4) lowest, heaviest stained doublet (d)
MS samples were prepared using Stage Tips
DISCUSSION
anti-ORF2 (clone 9) gave high background for Western, should use clone 11 or clone 5 next time. Clone 5 seems to work best for Western