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Found 11 matching solutions for this experiment
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Immunoprecipitation (IP) and immunoblotting (IB) were performed as previously described [42]. In some cases, membranes were stripped and re-probed with specific antibodies. To quantify changes, the densitometries of protein bands were determined with a calibrated GS-670 densitometer. For endogenous interactions, HeLa cells grown in 10 cm2 dishes were harvested and the cell lysates were then subjected to IP. All IP and IB experiments were performed as three independent experiments. |
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| Protocol tips |
| Immunoprecipitation (IP) and immunoblotting (IB) were performed as previously described [42]. In some cases, membranes were stripped and re-probed with specific antibodies. To quantify changes, the densitometries of protein bands were determined with a calibrated GS-670 densitometer. For endogenous interactions, HeLa cells grown in 10 cm2 dishes were harvested and the cell lysates were then subjected to IP. All IP and IB experiments were performed as three independent experiments. |
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| - For Western Blot, lyse cells with RIPA buffer (50 mM Tris, 1.0 mM EDTA, 150 mM NaCl, 0.1% Triton X-100, 1% sodium deoxycholate, 1 mM PMSF) |
- For Western Blot dilute primary Ab at 1:1000 and incubate at 4 °C overnight.
- For IF, dilute primary Ab between 1:500-:1000 and incubate at 4 °C overnight. |
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| Upstream tips |
| - For Western Blot, lyse cells with RIPA buffer (50 mM Tris, 1.0 mM EDTA, 150 mM NaCl, 0.1% Triton X-100, 1% sodium deoxycholate, 1 mM PMSF) |
| Protocol tips |
- For Western Blot dilute primary Ab at 1:1000 and incubate at 4 °C overnight.
- For IF, dilute primary Ab between 1:500-:1000 and incubate at 4 °C overnight. |
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| - Lyse cells in 4X Laemmli buffer (0.125 M tris-HCl, pH 6.8, 4% SDS, 0.13mM bromophenol blue, 1 M sucrose) containing 0.5% ß mercaptoethanol |
- Dilute primary Ab at 1:1000
- Incubate primary antibody for 16 hours |
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| Upstream tips |
| - Lyse cells in 4X Laemmli buffer (0.125 M tris-HCl, pH 6.8, 4% SDS, 0.13mM bromophenol blue, 1 M sucrose) containing 0.5% ß mercaptoethanol |
| Protocol tips |
- Dilute primary Ab at 1:1000
- Incubate primary antibody for 16 hours |
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| - Lyse cells in RIPA buffer (50 mM Tris, 1.0 mM EDTA, 150 mM NaCl, 0.1% Triton X-100, 1% sodium deoxycholate, 1 mM PMSF) |
-Dilute primary Ab at 1:1000 and at 4 °C overnight, |
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| Upstream tips |
| - Lyse cells in RIPA buffer (50 mM Tris, 1.0 mM EDTA, 150 mM NaCl, 0.1% Triton X-100, 1% sodium deoxycholate, 1 mM PMSF) |
| Protocol tips |
| -Dilute primary Ab at 1:1000 and at 4 °C overnight, |
| Upstream tips |
Protocol tips |
Downstream tips |
| - Lyse cells in RIPA buffer (50 mM Tris, 1.0 mM EDTA, 150 mM NaCl, 0.1% Triton X-100, 1% sodium deoxycholate, 1 mM PMSF) |
- Incubate primary antibody at 4 °C overnight |
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| Upstream tips |
| - Lyse cells in RIPA buffer (50 mM Tris, 1.0 mM EDTA, 150 mM NaCl, 0.1% Triton X-100, 1% sodium deoxycholate, 1 mM PMSF) |
| Protocol tips |
| - Incubate primary antibody at 4 °C overnight |
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| - Lyse cells in RIPA buffer that contained 1% protease and phosphatase inhibitors |
- Dilute Ab at 1:1000
- Incubate primary antibody overnight at 4°C. |
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| Upstream tips |
| - Lyse cells in RIPA buffer that contained 1% protease and phosphatase inhibitors |
| Protocol tips |
- Dilute Ab at 1:1000
- Incubate primary antibody overnight at 4°C. |
| Upstream tips |
Protocol tips |
Downstream tips |
| - Lyse cells in RIPA buffer containing protease and phosphatase inhibitors |
- Dilute primary Ab between
1:500-1:3000 and incubate overnight at 4 °C. |
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| Upstream tips |
| - Lyse cells in RIPA buffer containing protease and phosphatase inhibitors |
| Protocol tips |
- Dilute primary Ab between
1:500-1:3000 and incubate overnight at 4 °C. |
| Upstream tips |
Protocol tips |
Downstream tips |
| - Lyse cells with RIPA buffer that contained 1% protease and phosphatase inhibitors. |
- Incubate primary antibody overnight at 4°C. |
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| Upstream tips |
| - Lyse cells with RIPA buffer that contained 1% protease and phosphatase inhibitors. |
| Protocol tips |
| - Incubate primary antibody overnight at 4°C. |
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| - Seed 2 × 10^4 cells/well |
- Add reagent and incubate the cells at 37 °C with 5% CO2 for 15 minutes to 1 hour |
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| - Seed 2 × 10^4 cells/well |
| Protocol tips |
| - Add reagent and incubate the cells at 37 °C with 5% CO2 for 15 minutes to 1 hour |
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- Dilute primary Ab between 1:100-1:1000
- Incubate primary antibody at 8°C overnight |
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| Protocol tips |
- Dilute primary Ab between 1:100-1:1000
- Incubate primary antibody at 8°C overnight |
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- Dilute primary Ab at 1:100
- Incubate primary antibody at 8°C overnight |
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| Protocol tips |
- Dilute primary Ab at 1:100
- Incubate primary antibody at 8°C overnight |
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