Difference between revisions of "Team:Pasteur Paris/Immunology"
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<U>Protocol: </U></br></br> | <U>Protocol: </U></br></br> | ||
<strong><U>Sample preparation:</U> </strong></br> | <strong><U>Sample preparation:</U> </strong></br> | ||
| − | The sample is tagged using EZ-Link kit </br></br> | + | The sample is tagged using EZ-Link kit. </br></br> |
1. Add 500mL of ultrapure water to the dry-blend Phosphate Buffered Saline (PBS).</br></br> | 1. Add 500mL of ultrapure water to the dry-blend Phosphate Buffered Saline (PBS).</br></br> | ||
| − | 2. Prepare 1mg of antibody in 1.0ml of PBS(+/-BSA1%)</br></br> | + | 2. Prepare 1mg of antibody in 1.0ml of PBS(+/-BSA1%).</br></br> |
3. Reconstitute 1mg of lyophilized EZ-Link Plus Activated Peroxidase with 100 µl of ultrapure water and add it to the antibody solution or add the protein sample directly to the lyophilized activated. </br></br> | 3. Reconstitute 1mg of lyophilized EZ-Link Plus Activated Peroxidase with 100 µl of ultrapure water and add it to the antibody solution or add the protein sample directly to the lyophilized activated. </br></br> | ||
| − | 4. In a fume hood, immediately add 10 µl of Sodium Cyanoborohydride to the reaction and incubate for 1 hour at room temperature </br></br> | + | 4. In a fume hood, immediately add 10 µl of Sodium Cyanoborohydride to the reaction and incubate for 1 hour at room temperature. </br></br> |
5. Add 20 µl of Quenching Buffer and react at room temperature for 15 minutes. </br> </br> | 5. Add 20 µl of Quenching Buffer and react at room temperature for 15 minutes. </br> </br> | ||
| − | <i>The conjugate can be store at 4°C for up to 4 weeks. </i></br> </br> </br> | + | <i> → The conjugate can be store at 4°C for up to 4 weeks. </i></br> </br> </br> |
<strong><U>Membrane:</U> </strong></br> | <strong><U>Membrane:</U> </strong></br> | ||
| − | 1. <strong>Coating:</strong> put 1 µl of a non-diluted antibody on the membrane, let the membrane dry for 5 minutes at room temperature </br> </br> | + | 1. <strong>Coating:</strong> put 1 µl of a non-diluted antibody on the membrane, let the membrane dry for 5 minutes at room temperature. </br> </br> |
2. <strong>Saturation:</strong> </br> | 2. <strong>Saturation:</strong> </br> | ||
| − | • Put the membrane in PBS-Tween 0,5%-milk 5% on a rocker, 1 hour at room temperature </br> | + | • Put the membrane in PBS-Tween 0,5%-milk 5% on a rocker, 1 hour at room temperature. </br> |
| − | • Replace the washing solution with a fresh one </br> </br> | + | • Replace the washing solution with a fresh one. </br> </br> |
| − | 3. <strong>Binding:</strong> Add the sample in the PBS-Tween 0,5%-milk 5% solution at the appropriate dilution, incubate overnight at 4°C on a rocker </br> </br> | + | 3. <strong>Binding:</strong> Add the sample in the PBS-Tween 0,5%-milk 5% solution at the appropriate dilution, incubate overnight at 4°C on a rocker. </br> </br> |
| − | 4. <strong>Washing:</strong> Wash the membrane 3 times for 5 minutes on a rocker at room temperature </br> </br> | + | 4. <strong>Washing:</strong> Wash the membrane 3 times for 5 minutes on a rocker at room temperature. </br> </br> |
| − | 5. <strong>Revelation:</strong> Reveal the membrane using approximately 1ml of Pierce ECL Western Blotting substrate </br> </br> | + | 5. <strong>Revelation:</strong> Reveal the membrane using approximately 1ml of Pierce ECL Western Blotting substrate. </br> </br> |
| − | To test the technic, we used our protocol and checked that we were able to detect pure viral protein bind to EZ-link. We used the previous protocol, on the membrane, we coated 4G2, BSA was used as negative control. On the membranes, we incubate the membrane with the following solution. Negative control are membrane A to D, to validate the experiment, membrane E has to be positive </br> </br> | + | To test the technic, we used our protocol and checked that we were able to detect pure viral protein bind to EZ-link. We used the previous protocol, on the membrane, we coated 4G2, BSA was used as negative control. On the membranes, we incubate the membrane with the following solution. Negative control are membrane A to D, to validate the experiment, membrane E has to be positive. </br> </br> |
• A: YFV E-protein + HRP EZ-link </br> | • A: YFV E-protein + HRP EZ-link </br> | ||
• B: YFV E-protein only </br> | • B: YFV E-protein only </br> | ||
