Test PO with new CDI beads
Date: 07/20/17
Cell lines:
HEK293 FLp-In T-Rex (4/5/17)
HeLa Kyoto (8/11/16)
Beads made on 7/18/17
| Number | Antibody | Clone ID | Expression | Cell line used for Testing |
|---|---|---|---|---|
| 1 | E2F4 | YP917.1.3C9 | HeLa, colon tumor, HEK | HeLa |
| 2 | FOXC2 | YP828.1.1B1 | HeLa, colon tumor | HeLa |
| 3 | ATF2* | YP185.1.1B4 | HeLa, HEK | HeLa |
| 4 | NR3C2 | YP596.1.1E3 | HEK, colon tumor | HEK |
| 5 | DACH1 | R1477.1.1C9 | HEK, colon tumor, HeLa | HEK |
| 6 | USF1 | YP605.1.1C10 | HeLa, HEK, colon tumor | HeLa |
*ATF2 - I made an error on this TF; I thought it was aka NFKB-p65, (RELA is actually aka NFKB-p65). However, based on WB results, it seems that either anti-p65 binds ATF2, or ATF2 interacts with p-65 and the anti-p65 binds the complex.
Extraction buffers:
20mM HEPES, pH7.4, 0.5% Tritonx-100, 150mM NaCl
20mM HEPES, pH7.4, 0.5% Tritonx-100, 300mM NaCl
Weigh out 8x 100mg of HeLa powder and 4x 100mg of HEK powder; add 400ul of extraction buffer to each tube (1:4 w/v), vortex to mix
Sonicate 25x 2 sec at 2 Amp (same as TP53 sonication regime, Energy output: HeLa samples ~100J; HEK samples ~70J?)
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Combine the supernatant from the same buffer condition, save 20ul for Western (Input, load 2ul)
Split the rest of each into 4 equal parts
Set up the 100mg IP reactions
Incubate @ 4°C for 30’
After IP, save 5ul of each flow-through for Western (FT, load 2ul)
wash 3x 1ml extraction buffer
Switch beads to fresh tubes at 2nd wash step
Elute with 18ul of LDS @ 70°C for 5' (E)
(We can try eluting @ RT if we see IgG H and L chairs cross the board on Coomassie gel)
Add DTT to 25mM to all samples and heat @75 °C for 10’; Save 10% of elution for Western
Chill the samples on ice
Add 100mM IOA to all samples; incubate in dark for 30’ @ RT
Run 4-12% Bis-Tris gels
Gel 1:15-well, Coomassie stain (90% of the elution)
- Marker
- E2F4_HeLa_150 (~44kD)
- E2F4_HeLa_300
- FOXC2_HeLa_150 (~54kD)
- FOXC2_HeLa_300
- ATF2_HeLa_150 (~55kD)
- ATF2_HeLa_300
- NR3C2_HEK_150 (~107kD)
- NR3C2_HEK_300
- DACH1_HEK_150 (~79kD)
- DACH1_HEK_300
- USF1_HeLa_150 (~34kD)
- USF1_HeLa_300
- BSA_50ng
- BSA_150ng
Gel 2: 26-well E2F4, FOXC2 and ATF2 Western (2ul of input or FT, 10% of the elution)
- Marker_5ul
- E2F4_Input_150 (HeLa)
- E2F4_FT_150
- E2F4_Input_300 (HeLa)
- E2F4_FT_300
- E2F4_E_150
- E2F4_E_300
- Marker_2ul
- Marker_5ul
- FOXC2_Input_150 (HeLa)
- FOXC2_FT_150
- FOXC2_Input_300 (HeLa)
- FOXC2_FT_300
- FOXC2_E_150
- FOXC2_E_300
- Marker_2ul
- Marker_5ul
- ATF2_Input_150 (HeLa)
- ATF2_Input_300
- ATF2_FT_150
- ATF2_FT_300
- ATF2_E_150
- ATF2_E_300
- Marker_2ul
Gel 2: 26-well, NR3C2, DACH1 and USF1 Western (2ul of input or FT, 10% of the elution)
- Marker_5ul
- NR3C2_Input_150 (HeLa)
- NR3C2_FT_150
- NR3C2_Input_300 (HeLa)
- NR3C2_FT_300
- NR3C2_E_150
- NR3C2_E_300
- Marker_2ul
- Marker_5ul
- DACH1_Input_150 (HeLa)
- DACH1_FT_150
- DACH1_Input_300 (HeLa)
- DACH1_FT_300
- DACH1_E_150
- DACH1_E_300 (This sample vol. was 40ul for some reason; put it in the speed vac to reduce the vol. to 20ul)
- Marker_2ul
- Marker_5ul
- USF1_Input_150 (HeLa)
- USF1_Input_300
- USF1_FT_150
- USF1_FT_300
- USF1_E_150
- USF1_E_300
- Marker_2ul
Wet transfer, 70V for 1.5hr
After transfer, cut between the two adjacent marker lanes
Blocking: TBST/5% milk, RT, 2hrs
Anti-E2F4 (Santa Cruz)
Primary Ab: mouse anti-E2F4 antibody, E2F-4 (D-7): sc-398543, starting @ 1:500 (1:100 to 1:1000) (4°C overnight)
Secondary Ab: HRP linked anti-mouse IgG, 1:10,000 (RT for 1hr)
Anti-FOXC2 (Santa Cruz)
Primary Ab: mouse anti-FOXC2 antibody, FOXC2 (G7) sc-515234, starting @ 1:500 (1:100 to 1:1000) (4°C overnight)
Secondary Ab: HRP linked anti-mouse IgG, 1:10,000 (RT for 1hr)
Anti-ATF2 (NFKB-p65, Bilogend) (test this one first)
Primary Ab: mouse anti-NF-κB p65 antibody, clone 14G10A21 (Biolegend Cat # 653001, starting @ 1:500 (1:500 to 1:5,000) (4°C overnight)
Antibody concentration 0.5 mg/ml, for Western use 0.1-1.0 µg per ml.
Secondary Ab: HRP linked anti-mouse IgG, 1:10,000 (RT for 1hr)
Also available Anti-ATF2 (Santa Cruz)
Primary Ab: mouse anti-ATF2 antibody, ATF-2 (F2BR-1) sc-242, starting @ 1:500 (1:100 to 1:1000) (4°C overnight)
Secondary Ab: HRP linked anti-mouse IgG, 1:10,000 (RT for 1hr)
anti-E2F4, FOXC2, ATF2 Western result
2-min exposure, high sensitivity, auto tone (longer exposure showed same result)
Anti-NR3C2 (Santa Cruz)
Primary Ab: mouse anti-NR3C2 antibody, MCR(H10 E4C9F) sc-53000, starting @ 1:500 (1:100 to 1:1000) (4°C overnight)
Secondary Ab: HRP linked anti-mouse IgG, 1:10,000 (RT for 1hr)
Anti-DACH1 (Santa Cruz)
Primary Ab: mouse anti-DACH1 antibody, DACH1 (A-6) sc-398706, starting @ 1:500 (1:100 to 1:1000) (4°C overnight)
Secondary Ab: HRP linked anti-mouse IgG, 1:10,000 (RT for 1hr)
Anti-USF1 (abcam)
Primary Ab: rabbit anti-USF1 antibody [EPR6430] (ab125020), starting @ 1:2,500 (1/10000 - 1/50000) (4°C overnight)
Secondary Ab: HRP linked anti-rabbit IgG, 1:10,000 (RT for 1hr)
anti-NR3C2, DACH1, USF1 Western result
2-min exposure, high sensitivity, auto tone (longer exposure show the same result)
