Difference between revisions of "Team:UCopenhagen/Protocols Lipid Bilayers"
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<p>The dye Atto655 is included in some of the lipid mixtures to enable detection of the lipid bilayers and liposome by fluorescence microscopy. Biotin was also included in some lipid mixtures to allow for attachment of biotin-containing liposomes to microscopy slides covered with NeutrAvidin.</p> | <p>The dye Atto655 is included in some of the lipid mixtures to enable detection of the lipid bilayers and liposome by fluorescence microscopy. Biotin was also included in some lipid mixtures to allow for attachment of biotin-containing liposomes to microscopy slides covered with NeutrAvidin.</p> | ||
| + | <table> | ||
| + | <tr> | ||
<td><strong>Lipid</strong> | <td><strong>Lipid</strong> | ||
| − | |||
</td> | </td> | ||
<td><strong>MW (g/mol)</strong> | <td><strong>MW (g/mol)</strong> | ||
| − | |||
</td> | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
<td>DOPC | <td>DOPC | ||
| − | |||
</td> | </td> | ||
<td>786.113 | <td>786.113 | ||
| − | |||
</td> | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
<td>DOPS | <td>DOPS | ||
| − | |||
</td> | </td> | ||
<td>810.025 | <td>810.025 | ||
| − | |||
</td> | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
<td>SM | <td>SM | ||
| − | |||
</td> | </td> | ||
<td>760.2 | <td>760.2 | ||
| − | |||
</td> | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
<td>Chl | <td>Chl | ||
| − | |||
</td> | </td> | ||
<td>386.7 | <td>386.7 | ||
| − | |||
</td> | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
<td>Atto655 | <td>Atto655 | ||
| − | |||
</td> | </td> | ||
<td>1366.0 | <td>1366.0 | ||
| − | |||
</td> | </td> | ||
| + | </tr> | ||
| + | <tr> | ||
<td>Biotin DSPE PEG (2000) | <td>Biotin DSPE PEG (2000) | ||
| − | |||
</td> | </td> | ||
<td>3016.0 | <td>3016.0 | ||
| − | |||
</td> | </td> | ||
| + | </tr> | ||
| + | </table> | ||
| + | |||
<p> Lipids solubilized in chloroform and methanol should be handled in a fume hood and pipetted using a Hamilton syringe. </p> | <p> Lipids solubilized in chloroform and methanol should be handled in a fume hood and pipetted using a Hamilton syringe. </p> | ||
<h3>Materials</h3> | <h3>Materials</h3> | ||
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<li>Biotin, PEG, 0.5 mg/mL (referred to as "stock 1" in notebook) | <li>Biotin, PEG, 0.5 mg/mL (referred to as "stock 1" in notebook) | ||
<li>Chloroform and methanol** | <li>Chloroform and methanol** | ||
| + | |||
| + | <p>*Lipids: SM, DOPC, DOPS and Chl. </p> | ||
| + | <p>**A mix of chloroform and methanol is used for DOPC and SM solutions and chloroform alone for the rest of the lipid solutions.</p> | ||
| + | <h3>Procedure</h3> | ||
| + | <p> Dilute 25 mg/mL lipid stocks to new stocks with concentrations of 1 μg/μL (referred to as “stock 2” in notebook).</p> | ||
| + | <p>Dilute biotin and dye to stocks of 0.1 μg/μL and 0.1 μg/μL (referred to as “stock 2” in notebook).</p> | ||
| + | <p>Make lipid mastermixes 1, 2 and 3 from the stocks 2s by adding the following.</p> | ||
| + | |||
| + | |||
| + | |||
| + | |||
| + | |||
<h1>Overview of SLB protocols</h1> | <h1>Overview of SLB protocols</h1> | ||
Revision as of 10:34, 16 October 2018
Protocol: supported lipid bilayer experiments
iGEM UCopenhagen 2018
Lipid film stocks
Lipid mixtures containing sphingomyelin (SM), dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidylserine (DOPS), and cholesterol (Chl) in a ratio of 44:24:12:20 (SM:DOPC:DOPS:Chl) is prepared for subsequent use to make supported lipid bilayer and liposomes. This composition is inspired from Chatterjee and colleagues [1].
The dye Atto655 is included in some of the lipid mixtures to enable detection of the lipid bilayers and liposome by fluorescence microscopy. Biotin was also included in some lipid mixtures to allow for attachment of biotin-containing liposomes to microscopy slides covered with NeutrAvidin.
| Lipid | MW (g/mol) |
| DOPC | 786.113 |
| DOPS | 810.025 |
| SM | 760.2 |
| Chl | 386.7 |
| Atto655 | 1366.0 |
| Biotin DSPE PEG (2000) | 3016.0 |
Lipids solubilized in chloroform and methanol should be handled in a fume hood and pipetted using a Hamilton syringe.
Materials
- 25 mg/mL stocks of each lipid* (referred to as "stock 1" in notebook)
- Lipid dye Atto655, 0.5 mg/mL (referred to as "stock 1" in notebook)
- Biotin, PEG, 0.5 mg/mL (referred to as "stock 1" in notebook)
- Chloroform and methanol**
*Lipids: SM, DOPC, DOPS and Chl.
**A mix of chloroform and methanol is used for DOPC and SM solutions and chloroform alone for the rest of the lipid solutions.
Procedure
Dilute 25 mg/mL lipid stocks to new stocks with concentrations of 1 μg/μL (referred to as “stock 2” in notebook).
Dilute biotin and dye to stocks of 0.1 μg/μL and 0.1 μg/μL (referred to as “stock 2” in notebook).
Make lipid mastermixes 1, 2 and 3 from the stocks 2s by adding the following.
Overview of SLB protocols
An overview of the protocols used in the Supported Lipid Bilayers experiments.
Liposomes preparation step-by-step
Protocol from Nikos Hatzakis’ enzyme lab. Explains the process of making liposomes. When making liposomes in our own lab, we used liquid nitrogen instead of acetone and dry ice.
Liposome preparation
Excel sheet to calculate amount of lipid added when making liposomes. Goes with the protocol Liposomes preparation step-by-step.
Preparation of supported lipid bilayers
When making supported lipid bilayers it is based on this protocol step 8 to 12.
Supported lipid bilayers experiments
Internal protocol for the experiments performed in the confocal microscope with supported lipid bilayers. The protocol contains parts from other external protocols.