Two Approcahes To Studying Cell Membranes

1) Microscopy
a. Electron Microscopy
b. Light Microscopy
i. Fluorescence Microscopy
2) Membrane Fractionation
In Electron Microscopy you take a piece of cell/tissue and use a
chemical to crosslink the molecules in the cell/tissue (to avoid their
floating around). Remove water by dehydrative steps, and then embed
them in a plastic resin (to make a plastic block). This preserves the
molecular structure of the membrane (despite the specimen being
dead). Subject it to very thin slices into the cell (with diamond cutting
tool) at around 100 nm thickness. Then you stain the specimen on a
ribbon (that attach to parts of the cell) and put it into an electron
microscope. The electron beam is fired onto the specimen that the
stain scatters and you can observe the reflection of the scattered
electrons.
Light Microscopy is where there have been recent advances, and is
where the future of microscopy may lie. Fluorescent microscopy
involves a green pigment isolated from a jellyfish (Green Fluorescent
Pigment - GFP). You isolate the gene from GFP and use it to append it
to any other protein that would carry the GFP molecule with it allowing
the observation of where it is in the cell. Its like you tag a protein
visually and can watch it in real time. (On a side note the person who
found the technique is now a truck driver and people who picked up his
work won a Nobel Prize). Of course, fusing GFP with a protein can
cause it to behave differently.

In Membrane Fractionation you take tissue and subject the tissue to a

technique that ruptures the cell (usually intense homogenization). The
process involves putting the tissue in a tube and then sticking a tightly

fitting (usually Teflon) pestle inside and back and forth. This gives you
a lysate containing many membrane fragments (e.g. plasma
membrane, endoplasmic reticulum, nuclear membrane, mitochondrion
membrane, lysosome membrane, golgi apparatusetc). These are
floating around in organelles as a soup. You then can gently use a
centrifuge to spin it lightly, the first thing to fall out of suspension is
the nuclear membrane. On the other hand you need a higher level of
RPM to settle bubbles of membranes known as vesicles. You can of
course use differential centrifugation (spinning at various speeds).
Membranes largely consist of lipids, making it light. But pieces of the
ER contain ribosomes in the membrane (for secretory protein
production) have higher buoyant density. You can use a sucrose
gradient to help you isolate the high buoyant density particles from
the low buoyant density ones. To do this you just create a gradient with
the higher concentration of sucrose at the bottom of the tube and then
pour the lysate soup at the top and put it in a centrifuge until it
reaches equilibrium. This helps as the higher buoyant density particles
will be at the bottom of the tube. The two processes of sucrose
gradient and differential centrifugation can leave you with the ability to
separate two quite homogenous types of membranes.

Properties of Membranes
Membranes are Selectively Permeable
They expel charged particles/polar particles/expel sodium ions
etc. Hundreds or thousands of permeases or transport channels
allow the cell to perform these tasks. Membranes alone are generally
impermeable to ionic/charged particles.
Also theres oxidative phosphorylation in the mitochondrial
envelope, in fact its possible that mitochondria evolved from some
sort of symbiosis of eukaryotic cells and primitive bacterial cells. The
outer membrane has stable channels known as porins that are
essentially just pores, theyre selective only by size (~100 daltons) and

are a method of passive transport. The inner membrane is very active

in the transport of charged particles (the stuff inside the inner
membrane is known as the matrix). When theres an excess of
proteins on the outside of the mitochondrial matrix, they drive the ATP
synthase (a protein on the surface of the mitochondrial envelope) to
produce ATP. This would be intrinsically impossible if the surface of
the membrane was permeable naturally.