The LPI™ FlowCell Kits are used for immobilization of membrane proteins in their native membranes. By extracting the desired cell membrane and creating vesicles (proteoliposomes) the functional proteins can be attached to a solid surface and then subjected to various treatments. Since the proteins are not extracted from the membrane and the proteoliposomes are hard to detach from the surface, this technology does not allow for membrane protein purification in that sense.
The binding of proteoliposomes involves a strong interaction of both lipid and protein to the surface.
We recommend at least 10x106 cells as starting material. This amount allows for several LPI™ FlowCell runs. The binding capacity of one FlowCell is approximately 100 µg of proteoliposomes.
The volume of LPI™ Maxi FlowCell is 350 µl ï¿½l and 40 µl of LPI™ HexaLane FlowCell.
Since the binding of proteoliposomes to the LPI™ FlowCell surface is very strong, this does not allow for reusability. Most of the material can be removed with various protocols such as use of detergents, but since we cannot guarantee 100% removal, we do not recommend reusing the FlowCell. One LPI™ FlowCell provides enough peptides for several mass spectrometry runs, depending on the injection volume and sample capacity of the analysis system.
The starting material for making vesicles is a membrane preparation from the cells of interest. The large membrane sheets are normally broken down by extrusion or sonication, where tip sonication is the recommended method. The membrane preparation is subjected to a tip sonication protocol that results in a solution of vesicles, 50-150 nm in diameter. This solution is injected into LPI™ FlowCell.
We have successfully used the following cell types: E. Coli bacteria, mast cells, insect cells, blood cells, Jurkat cells, anammox bacteria, skeletal muscle tissue, stem cells, breast carcinoma cells, Arabidopsis thaliana, Helicobacter pylori. Membrane samples from all cell types tested thus far have been successfully immobilized on the FlowCell surface.
Yes, if at least 100 µg membrane material can be extracted from the tissue sample at a concentration of 0.1-1 mg/ml.
It should be possible to attach whole cells to the surfaces of LPI™ FlowCell. The attached cells may however be affected by the flow when washing the flow cell and adding reagents. A low flow rate through the flow cell is recommended. The spacing between the substrates is approximately 50 µm inside the LPI™ FlowCell and this sets a size limit on the cells applicable to the flow cell.
There is currently no application data for ligand-binding studies. In general, binding of target preparations to the surfaces of LPI™ FlowCell followed by addition of ligands should be possible. Specific protocols and evaluation methods must be developed and optimized in each case and we encourage these kinds of collaborations with our customers.
Prior to LPI™ FlowCell the cells need to be broken and extracted. This usually requires a homogeniser, such as Dounce, and an ultra centrifuge. A tip sonicator such as Vibra Cell (model 501) from Sonics & Materials Inc. is used to disrupt the large membrane sheets into smaller pieces to get a small and homogenous size of the proteoliposomes (50-150 nm in diameter).
The eluted peptide sample is usually enough for at least three mass spectrometry runs. The exact number depends on several factors such as injection volume and the sample capacity of the LC-MS system for optimal resolution, which differ between different LC-MS set-ups.
A general membrane preparation protocol is available (click on Protocols and MSDS). Please contact us if you have specific questions.
The diameter size of the proteoliposomes is recommended to be 50-150 nm; once immobilized, vesicles this size are not affected by the flow of reagents through the FlowCell.
When cells are lysed, the result is large membrane sheets. These sheets are then fragmented during tip-sonication (or extrusion) to create membrane vesicles, also called proteoliposomes, meaning small lipid vesicles containing membrane proteins.