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FAQ
Can I use the LPI™ SamplePrep kit for
membrane protein purification?
The LPI SamplePrep kit is 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.
What kind of interaction takes place between the proteoliposomes
and the coated surface?
The binding of proteoliposomes involves a strong interaction of
both lipid and protein to the surface.
How much starting material do I need?
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.
What is the volume of LPI FlowCell?
The volume of LPI FlowCell is 350 µl.
Can I reuse your product?
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.
How do I prepare the membrane vesicles?
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.
What kind of cells can be used with the 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. Membrane samples from all cell types tested thus
far have been successfully immobilized on the FlowCell surface.
Can I use tissue as starting material?
Yes, if at least 100 µg membrane material can be extracted from
the tissue sample at a concentration of 0.1-1 mg/ml.
Can I apply whole cells to LPI FlowCell?
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.
Can I use LPI SamplePrep Kit for ligand-binding applications?
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.
What other equipment is needed prior to the use of LPI FlowCell?
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).
How many MS analyses can the eluted peptides from one LPI
FlowCell be used for?
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.
Do you have protocols for the preparation of vesicles from my
cell line?
A general membrane preparation protocol is available (click on
Protocols and MSDS). Please contact us if you have specific
questions.
What should the size of the proteoliposomes be prior to loading
on LPI FlowCell?
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.
Does "proteoliposome" mean the fragment of the cell membrane? Are
"vesicle" and "proteoliposome" the same?
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.
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