Biology Reference
In-Depth Information
strength of the buffer. Ficoll is extensively used in density gradient centrifugations (discussed
below). In addition to the osmoticum (e.g. Ficoll/sucrose), many additional compounds are
included in membrane isolation buffers. These include pH buffers, chelators to remove harm-
ful divalent metals, antioxidants, and inhibitors of many degradative enzymes. Homogeni-
zation buffers are indeed a complex 'soup'.
The investigator must have many tools in his bag and work quickly, to identify and sepa-
rate the various membrane fractions before serious artifacts arise. It is impossible to obtain an
absolutely pure membrane; at best just an enriched fraction can be isolated.
Membrane Isolation Steps
The general sequence of steps involved in membrane isolation includes
[5,6,8]
:
Identify the best cell to obtain the membrane of interest.
Isolate and clean the cells.
Break open the cell (homogenization).
Separate large intact organelles (fractionation).
Separate the small microsomal cell fractions.
Identify the cell fractions by use of appropriate membrane markers (both positive and
negative markers).
Possible additional steps may include:
Isolate and purify a specific membrane protein.
Isolate and purify membrane lipids.
Reconstitute the membrane protein into lipid vesicles.
Determine the mechanism of action of the reconstituted protein.
B.
BREAKING OPEN THE CELL: HOMOGENIZATI
ON
With the final objective in mind, the proper cell type is chosen and cleaned of unrelated
debris such as veins. If the starting tissue is solid (e.g. liver, muscle etc), the target cells
must be separated from neighboring cells. This process may involve use of proteases or other
enzymes that disaggregate adjacent cells and may be aided by chelating agents that bind
divalent cations. Disaggregation will alter some physical structures that reside on the cell
plasma membrane outer surface (e.g. gap junctions and tight junctions). Obviously if the
objective is to isolate and reconstitute rhodopsin for light receptor studies, liver, brain, and
muscle tissue would be inappropriate. Once sufficient quantities of the cell are obtained,
the cell must be broken open by as gentle a method as possible. This process is referred to
as 'homogenization'
[2,5,6,8]
. Vigorous procedures result in damaged membranes and
produce many small vesicular microsomes that are difficult to separate from one another.
If the procedure is too gentle, many cells will remain intact. Since it is estimated that even
the simplest, routine membrane isolation procedures take ~15% of an investigator's time
[10]
, a variety of cell disruption methodologies have been developed for specific applications.
For a brief comparison of the advantages and disadvantages of many of the methods dis-
cussed below see reference
[11]
.
