His-FLAG-SUMO targets pull down in denaturing conditions
Lysis:
1. Grow 10 ml of cells ON.
2. Dilute in 1L and grow until OD600=0.6/0.7. Then treat cells with 200 nM rapamycine for 30 min.
3. Harvest cells, wash with water, centrifuge and freeze down in liquid nitrogen (store @ -80)
4. Ressuspend the pellet in cold lysis buffer to a final volume of 25 ml.
5. After 20 min on ice, add 25 ml of cold 55% TCA and incubate 20 min on ice.
6. Pellet proteins by centrifugation at 2500g, 4°C for 20 min.
7. Wash the pellet with ice cold water and ressuspend in 25 ml of buffer A + 0.05% Tween20.
8. Transfer to centrifuge tubes and incubate 1 h at RT under agitation for solubilization.
9. Centrifuge 20 min, 4°C, 15000g and transfer SNT to new tube.
His-FLAG-SUMO pull-down (NiNTA):
1. Add imidazole to a final concentration of 20 mM.
2. Add 0.35 ml of NiNTA agarose resin and incubate ON at 4°C.
3. Pour the resine into a column and wash with 7 ml of buffer A + 0.05% Tween20.
4. Wash with 15 ml of buffer C + 0.05% Tween20 (@RT).
5. Elute 4 times with 0.2 ml of buffer C + 250 mM imidazole and pool the 4 fractions.
6. Add 1 vol of TCA 55% and incubate 20 min on ice.
7. Pellet proteins by centrifugation at 2500g, 4°C for 20 min.
8. Wash the pellet (invisible sometimes) with water and centrifuge.
9. Add 25 μl of buffer HU + 10 μl of water and heat 10 min at 65°C.
10. Add 6μl of loading buffer, SDS-PAGE (TGX gels), coomassie staining (R250).
Buffers:
Lysis buffer: 1.85 M NaOH
7.5% β-ME
Buffer A: 6 M guanidine-HCl
100 mM NaH2PO4
10 mM Tris/HCl
Adjust pH to 8.0 with NaOH
Buffer C: 8 M urea
100 mM NaH2PO4
10 mM Tris/HCl
Adjust pH to 6.3 with HCl
HU Buffer: 8 M urea
5% SDS
200 mM Tris/HCl, pH 6.8
1.5% DTT
RIPA buffer: 50 mM Tris/HCl, pH 7.4
150 mM NaCl
1% triton-X100
0.1% SDS
0.5% DOC
1. Grow 10 ml of cells ON.
2. Dilute in 1L and grow until OD600=0.6/0.7. Then treat cells with 200 nM rapamycine for 30 min.
3. Harvest cells, wash with water, centrifuge and freeze down in liquid nitrogen (store @ -80)
4. Ressuspend the pellet in cold lysis buffer to a final volume of 25 ml.
5. After 20 min on ice, add 25 ml of cold 55% TCA and incubate 20 min on ice.
6. Pellet proteins by centrifugation at 2500g, 4°C for 20 min.
7. Wash the pellet with ice cold water and ressuspend in 25 ml of buffer A + 0.05% Tween20.
8. Transfer to centrifuge tubes and incubate 1 h at RT under agitation for solubilization.
9. Centrifuge 20 min, 4°C, 15000g and transfer SNT to new tube.
His-FLAG-SUMO pull-down (NiNTA):
1. Add imidazole to a final concentration of 20 mM.
2. Add 0.35 ml of NiNTA agarose resin and incubate ON at 4°C.
3. Pour the resine into a column and wash with 7 ml of buffer A + 0.05% Tween20.
4. Wash with 15 ml of buffer C + 0.05% Tween20 (@RT).
5. Elute 4 times with 0.2 ml of buffer C + 250 mM imidazole and pool the 4 fractions.
6. Add 1 vol of TCA 55% and incubate 20 min on ice.
7. Pellet proteins by centrifugation at 2500g, 4°C for 20 min.
8. Wash the pellet (invisible sometimes) with water and centrifuge.
9. Add 25 μl of buffer HU + 10 μl of water and heat 10 min at 65°C.
10. Add 6μl of loading buffer, SDS-PAGE (TGX gels), coomassie staining (R250).
Buffers:
Lysis buffer: 1.85 M NaOH
7.5% β-ME
Buffer A: 6 M guanidine-HCl
100 mM NaH2PO4
10 mM Tris/HCl
Adjust pH to 8.0 with NaOH
Buffer C: 8 M urea
100 mM NaH2PO4
10 mM Tris/HCl
Adjust pH to 6.3 with HCl
HU Buffer: 8 M urea
5% SDS
200 mM Tris/HCl, pH 6.8
1.5% DTT
RIPA buffer: 50 mM Tris/HCl, pH 7.4
150 mM NaCl
1% triton-X100
0.1% SDS
0.5% DOC