Anti-FOXC2 and ATF2 PO for MS (no Glutaraldehyde)
Date: 8/14/17-8/17/17
Cell line: HeLa Kyoto (8/11/16)
Beads: anti-FOXC2 (7/18/17, YP828.1.1B1); anti-ATF2 (7/18/17, YP185.1.1B4); mouse IgG (Sigma, cat # I5381)
Scale: 100mg per experiment, with 10ul of beads, 4 replicates per condition
Extraction/wash buffers:
20mM HEPES, pH7.4, 0.5% Tritonx-100, 150mM NaCl 20mM HEPES, pH7.4, 0.5% Tritonx-100, 300mM NaCl
Samples will be:
4x anti-FOXC2 @ 150mM NaCl 4x anti-ATF2 @ 150mM NaCl 4x mouse IgG @ 150mM NaCl 4x anti-FOXC2 @ 300mM NaCl 4x anti-ATF2 @ 300mM NaCl 4x mouse IgG @ 300mM NaCl
Weigh out 12x 100mg aliquots of HeLa powder. Do each set in a day
Add 400ul buffer with protease inhibitors to powder, mix by vortexing, try to let the powder resuspend completely – typically takes ~10 sec; no more than 30 sec. Do not let the tube come to room temperature.
Sonicate 25x 2 sec @ 2 Amp (energy output ~100J)
Spin @ 20k rcf, 4°C for 10’ (Eppendorf Centrifuge 5417R)
Save 20ul of the supernatant for Western analysis (“Sup”)
Store the pellets in -20°C
Do the following if we decide to do Western on the pellets: resuspend each pellet in 1.1x LDS using the same vol. as Sup recovered (Vol. Sup); Resuspend by vortexing
Incubate @70°C for 5’ with shaking Spin @ 20k rcf, 25°C for 5’ Keep the supernatant for Western analysis (“Pellet”) Hold on ice and freeze as soon as convenient.
Use the rest of the lysate to set up IPs using 10ul of beads per 100mg of IP
Incubate @ 4°C for 30’
Keep 20ul of the flow-through for Western analysis (“FT”);
Wash 3x 1ml extraction buffer (transfer the beads to fresh tubes during 2nd wash)
Elute with 20ul of LDS @ 70°C for 5’ with mixing
Collect the elution (“E”)
Add 1ul of 500mM DTT to each sample (final conc. 25mM)
Heat samples @ 70°C for 10’
Chill samples on ice
Add 2ul of 1M Iodoacetamide to each tube (final conc. 100mM)
Incubate in dark for 30’ @ RT
Save 5%(1ul) of each elution fraction for Western (“E”)
Input (Sup), FT, Pellet and Elution fractions will be saved for each replicate for Western. But western does not have to be done immediately, we will do it only if we need to.
Run 20% (4ul) of each sample on a gel for Silver stain and 75% (15ul) of each sample will be used for gel plug
Silver stain gel to check all replicates, (15-well 4-12% Bis-Tris gel)
Gel 1: 150mM NaCl
- Marker_0.5ul
- BSA_25ng
- 150_FOXC2-1
- 150_FOXC2-2
- 150_FOXC2-3
- 150_FOXC2-4
- 150_ATF2-1
- 150_ATF2-2
- 150_ATF2-3
- 150_ATF2-4
- 150_mouse IgG-1
- 150_mouse IgG-2
- 150_mouse IgG-3
- 150_mouse IgG-4
Gel 2: 300mM NaCl
- Marker_0.5ul
- Space
- 300_FOXC2-1
- 300_FOXC2-2
- 300_FOXC2-3
- 300_FOXC2-4
- 300_mouse IgG-1
- 300_mouse IgG-2
- 300_mouse IgG-3
- 300_mouse IgG-4
- 300_ATF2-1
- 300_ATF2-2
- 300_ATF2-3
- 300_ATF2-4
- BSA_25ng
Gel plugs for all replicates (26-well 4-12% Bis-Tris gel)
Gel 1: 150mM NaCl
- Marker_5ul
- Space
- 150_FOXC2-1
- 150_FOXC2-2
- 150_FOXC2-3
- 150_FOXC2-4
- Space
- 150_ATF2-1
- 150_ATF2-2
- 150_ATF2-3
- 150_ATF2-4
- Space
- 150_mouse IgG-1
- 150_mouse IgG-2
- 150_mouse IgG-3
- 150_mouse IgG-4
- Space
- BSA_150ng
Gel 2: 300mM NaCl
- Marker_5ul
- Space
- 300_FOXC2-1
- 300_FOXC2-2
- 300_FOXC2-3
- 300_FOXC2-4
- Space
- 300_ATF2-1
- 300_ATF2-2
- 300_ATF2-3
- 300_ATF2-4
- Space
- 300_mouse IgG-1
- 300_mouse IgG-2
- 300_mouse IgG-3
- 300_mouse IgG-4
- Space
- BSA_50ng
- BSA_150ng
- Space
- Marker_5ul
