DOTAP Liposomes Co-encapsulating NBD/Rhodamine
Description
Resonance Energy Transfer (RET) is the process in which one fluorophore, the energy donor, absorbs a photon and, thus, non-radiatively transfers excited state energy to another fluorophore, the energy acceptor. This phenomenon is caused by the overlapping of the emission energy band of the donor fluorophore with the excitation energy band of the acceptor fluorophore. The rate and efficiency of the energy transfer improves as the area of overlap between the energy bands increases. RET is also dependent on the inverse sixth power of the distance between the energy donor and energy acceptor. This process is utilized for detecting lipid mixing during membrane fusion. Membrane fusion is the process in which two clear lipid bilayers merge to then create one membrane. To detect lipid mixing, “labeled” liposomes are formulated with the fluorophores, while “unlabeled” liposomes are not. In order for detection to be successful, the assay calls for a very low concentration of the fluorophore donor/acceptor pair with the purpose of reducing membrane perturbation. As a result of this, the efficiency of the energy transfer decreases. A commonly used fluorophore pair for RET is N(7-nitrobenz-2-oxa-1,3-diazol-4-yl) phosphatidylethanolamine (N-NBD-PE) and N-(lissamine rhodamine B sulfonyl) PE (N-Rh-PE). Exchange between membranes is very minimal when using these probes, and because the fluorophore moiety is bonded to the head groups of PE, they do not cause any substantial perturbation of the membrane bilayer.
Technical Information
DOTAP Liposomes Co-encapsulating NBD/Rhodamine
For more information on the lipid composition of the liposomes mentioned above click here.
| Fusion assay liposomes (3 vial kit) | Specification |
|---|---|
| Vial 1 (Plain fusion liposomes) | This vial contains 2 ml of plain liposomes sized to 100 nm at total lipid concentration of 1 mM. |
| Vial 2 (fluorescent fusion liposomes) | This vial contains 2 ml of fluorescent liposomes sized to 100 nm at a total lipid concentration of 1 mM and containing 0.8 mol % of both fluorophores |
| Vial 3 (Calibrating liposomes) | This vial contains 2 ml of fluorescent liposomes sized to 100 nm at total lipid concentration of 1 mM and containing 0.08 mol % of both fluorophores |
| Fluorescent Dye | Excitation/Emission (nm) | Molecular Structure |
|---|---|---|
| 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (ammonium salt) (NBD on headgroup) | 460/535 |  |
| 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt)(Rhodamine lipid) | 560/583 |  |
| Buffer and Liposome Size for All Three Vials | Specification |
|---|---|
| Buffer | 10 mM TES containing 100 mM NaCl |
| pH | 7.4 |
| Liposome Size | 100 nm |
| Reaction Buffer (Buffer C) | Specification |
|---|---|
| Buffer | 10 mM TES containing 100 mM NaCl and 0.1 mM EDTA |
| pH | 7.4 |
Assay Protocol
- Mix aliquots of the labeled and unlabeled liposomes at a ratio of 1:9. For liposome suspension with total lipid concentration of 50 uM, this would be 5 uM of labeled fluorescent liposomes and 45 uM of plain unlabeled liposomes in 2 ml of Buffer C (buffer C will be provided with the kit).
- Set the excitation to 460 nm and the emission to 530 nm. Set the residual fluorescence of the mixture of fluorescent labeled liposomes and plain unlabeled liposomes to 0% fluorescent.
- Add 50 uM of calibrating liposomes (vial 3) to 2 ml of Buffer C. Calibrating liposomes AKA mock fused liposomes are used to set the fluorescence at 100% (maximum).
- Introduce the fusogen of interest and continuously monitor the fluorescence. The fusion of liposomes will result in an increase in NVD fluorescence as the fluorescent lipids dilute into the plain unlabeled liposomes.
- The percentage of lipid mixing as a function of time is given by the equation below where I(t) is the fluorescence intensity at time, t, I(0) is the residual fluorescence, and I(∞) is the maximum fluorescence from the calibrating liposomes.
M(t) = 100 x [I(t) – I(0)] / [I(∞) – I(0)]
Technical Notes
- Liposomes are unilamellar and sized to 100 nm. If you need them to be made in another size, then you should mention that at the time of ordering. Unilamellar liposomes can be made in 50 nm and 200 nm sizes. Liposomes that are larger than 200 nm have a multilamellar structure.
- Liposomes are made in PBS buffer at pH 7.4, but it can be made in any other buffer of your choice. You can specify your buffer at the time of ordering.
- Liposomes are made in degassed buffer that is purged with argon to avoid oxidation of the unsaturated phospholipids.
- Liposomes are made under sterile condition.
- Liposomes are unilamellar and, therefore, due to their small size, they will not settle in the bottom of the vial.
- If you need to take multiple aliquots out of the vial, for use over a period of time, then it is advised to take extreme care in not contaminating the vial. It is recommended to handle the vial under a sterile hood to maintain the sterility of the product.
- Liposomes should never be frozen. Ice crystals that form during freezing will rupture the lipid membrane of the liposomes and change the size the liposomes particles.
- Liposomes should be kept in a refrigerator at 4°C in order to avoid hydrolysis of the liposomes.
- Liposomes should be kept at 4°C and should NEVER be frozen.
Appearance
Liposome co-encapsulating NBD/Rhodamine formulation is a red translucent liquid. Usually due to the small size of liposomes no settling will occur in the bottom of the vial. The liposomes are packaged in an amber vial.
Ordering/Shipping Information
- All liposome based formulations are shipped on blue ice at 4°C in insulated packages using overnight shipping or international express shipping.
- Liposomes should NEVER be frozen. Ice crystals that form in the lipid membrane can rupture the membrane, change the size of the liposomes and cause the encapsulated drug to leak out. Liposomes in liquid form should always be kept in the refrigerator.
- Clients who order from outside of the United States of America are responsible for their government import taxes and customs paperwork. Encapsula NanoSciences is NOT responsible for importation fees to countries outside of the United States of America.
- We strongly encourage the clients in Japan, Korea, Taiwan and China to order via a distributor. Tough customs clearance regulations in these countries will cause delay in custom clearance of these perishable formulations if ordered directly through us. Distributors can easily clear the packages from customs. To see the list of the distributors click here.
- Clients ordering from universities and research institutes in Australia should keep in mind that the liposome formulations are made from synthetic material and the formulations do not require a “permit to import quarantine material”. Liposomes are NOT biological products.
- If you would like your institute’s FedEx or DHL account to be charged for shipping, then please provide the account number at the time of ordering.
- Encapsula NanoSciences has no control over delays due to inclement weather or customs clearance delays. You will receive a FedEx or DHL tracking number once your order is confirmed. Contact FedEx or DHL in advance and make sure that the paperwork for customs is done on time. All subsequent shipping inquiries should be directed to Federal Express or DHL.
Storage and Shelf Life
Storage
Liposomes co-encapsulating NBD/Rhodamine should always be stored at in the dark at 4°C, except when brought to room temperature for brief periods prior to animal dosing. DO NOT FREEZE. ENS is not responsible for results generated by frozen product.
Shelf Life
Liposomes co-encapsulating NBD/Rhodamine are made on daily basis. The batch that is shipped is manufactured on the same day. It is advised to use the products within 60 days of the manufacturing date.
