|  Cooking the starch gels.
Before we actually cook the gels, we have to prepare all of the
buffers and weigh out the potato
starch. Allozyme loci tend to be tissue specific. So even
though a loci may be found in many different tissues, it is usually
best expressed in a specific tissue which is run with a specific
buffer. The buffers are prepared in advance and in bulk. The tissues
most commonly used for analysis are muscle, heart, liver and eye.
Before the cooking process, we measure out the amount of buffer
to a proportional amount of potato starch. It has been found most
gels run best at a density of between 10% and 13% starch to buffer.
We heat up approximately 2/3 of the buffer to near the boiling
point. When the heated mixture is at the proper temperature, the
potato starch is added to the remaining cold buffer and mixed
with the boiling buffer solution. This is cooked over a gas stove
until the mix is bubbling and viscous. The hot gel mixture is
then degassed to remove all the air bubbles, as the bubbles interfere
with the migration of the enzymes, making the enzyme unable to
be scored.
 Pouring the gel into the gel frame.
Gel frames are assembled each day prior to pouring of the gel. The
gel frames consist of a glass plate for the bottom and plastic sticks,
which are about 1/3" thick, for the sides. The sticks are held
to the glass plate with clamps. The gel frames are measured out
to a specific size of either 4.5" or 5.25" wide, depending
of the gel being run. All gels are 9" long, which is long enough
to hold 50 samples per gel, separated with dye wicks. After the
gel has been degassed, and while it is still hot and viscous, it
is poured into the gel frames, making sure to completely fill the
frame. A plastic pipette is used to remove any remaining bubbles.
The object is to have the gel as pure as possible. After the gel
has cooled to the touch, it is wrapped in cling wrap to keep it
from drying out.
 Loading the starch gel.
The starch gel is loaded with up to
50 individual samples. Each wick is dipped into a test tube containing
supernatant from the following tissues
(muscle, liver, heart or eye) from one animal. The wick is then
placed at the origin (starting point or bottom) of the gel. The
upper portion of the gel is called
the anode and the lower, narrower portion is the cathode. Red
dye markers are placed between each group of 10 individuals for
a visual indicator so that the migration distance can be monitored.
When the gel is loaded and ready for pre-run, the anode and cathode
are pushed together creating contact with the wicks. Once the
pre-run (15 minutes) is complete, the wicks are pulled and the
gel is then ready for electrophoretic separation of the enzymes.
  A
starch gel "wired"for running.
The gels are hooked up to a power pack, which runs a current
through the gel. This current pushes the enzymes through the gel,
which separate out in the current to the enzyme patterns that
we score. The power packs are usually set at a power setting so
as not to cook the gel with too much current, but to push the
enzymes along over the course of 4-6 hours. The settings can run
from 150 to 450 volts and from 50 to 90 milliamps, depending on
the gel density and the speed at which we want the gel to run.
  Slicing and staining the gel.
Once the gel has migrated the proper distance, the gel is removed
from the frame and sliced into thin
slices for staining. The gel slices
are plated on plastic plates, and stained. The staining process
involves mixing various chemicals and reagents to bring out the
desired expression of the loci. The staining process was arrived
at through a process of chemistry and often of trial and error.
Over time, the basic recipe is tweaked, changed and tested to
come up with the best combination of chemicals. The mixed chemical
is mixed with agar, which is used to solidify up the stain so
it stays on the slice after it is poured. After the gel has been
stained it is placed into an incubator
for the final processing.
 Judy Berger removes stained gel from incubator.
Once the stain has solidified on the gel slice, we move the stained
gel to an incubator. The chemicals used in the staining process
are light sensitive, so we keep them in a dark incubator until
we are ready to score the gels. An incubator is used to speed
the chemical reaction of the stain. Once the stain has come up
enough to score, we photograph the stained gels for future reference.
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Questions and Answers
Q.Why
do you use potato starch?
A. Potato starch has been
found over years of trial and error to be the most effective medium
for allowing migration of the enzymes in a manner which allow
the gels to be easily scored. The potato starch is highly refined
to remove any impurities.
Q. What is a buffer and why is it used?
A. The buffers we use are
composed of many chemicals. Some of the common chemicals used
include: Citric Acid, Tris (Tris[hydroxymethyl]aminomethane),
EDTA (Etyhylenediamine-tetraacetic acid), Glycine, and Boric Acid.
There are others, but these are the main ones. We combine these
in many different combinations and pH's, each of which will allow
for a better expression of a particular enzyme.
Q.
What is a supernatant?
A. A portion of the tissue
sample is partially dissolved in a grinding buffer designed to
break open the cells of the tissue and place the enzymes into
suspension. The clear fluid above the tissue is called the supernatant?
Q.
How do you load a gel?
A. To load a gel, we cut the
gel into an anode and cathode. The cathode being the negative
end, is smaller and usually only 1" to 1 1/2" wide.
The anode can be from 3 1/2" to 4 1/2" wide. A wick
is soaked with tissue which is partially dissolved in a grinding
buffer designed to break open the cells of the tissue and put
the enzymes into a suspension. The tissues are first subsampled
into culture tubes (or test tubes). We buffer the tissue at this
time with the grinding buffer, and refreeze the tissue at -80
C. The process of adding grinding buffer and thawing and refreezing
the tissue will break apart the cells of the tissue making the
enzymes easier to go into solution and into the gel. After 50
of these tissue and buffer soaked wicks are placed into the gel,
the gel is hooked up to the current and prerun for 15 minutes.
After this prerun, the electricity is unhooked from the gel to
for wick removal and orgin cleaning. All wick and tissue debris
must be removed from the origin or the gel will run poorly. After
the 15 minute run, there is enough of the enzymes embedded in
the gel to give a good score. Once the wicks are pulled and the
origin cleaned, the gel is again hooked up to electricity and
run for the prescribed amount of time (up to 6 hours).
Q.
Why is the gel cut into two sections?
A. Just before we load the
gel with the tissue soaked wick, we cut the gel into an anode
and cathode. The anode, or positive end, of the gel is the widest
part of the gel. The cathode, or negative end, is usually only
1" or 1.5" in size. Enzymes in the anode migrate towards
the upper end of the gel with the positive current. The negative
enzymes migrate down the anode towards the bottom of the gel.
Both contain enzymes which we are interested in scoring, and often,
a loci will have elements which migrate both directions from the
origin, or cut we made in the gel. So when staining, we have to
match the two ends up for a proper scoring of the loci.
Q.
Can the enzyme paths be seen prior to staining.
A. Often we really don't know
how a gel ran until we stain the slice. We know from experience
that the enzyme will be expressed at a certain place on the gel
after the gel has run a prescribed amount of time but if the gel
was poured wrong, if the origin was dirty or somehow poorly run,
the gel will have a "glitch", and we will most likely
have to rerun that particular gel for that particular loci.
Q. Do the enzyme paths show
up immediately after staining?
A. Some do. In fact, we often
will not stain a slice for certain loci until the biologists are
ready to score that loci. If we stain it too far in advance, the
stain will come up so strong that it will over develop, doing
what we call "blowing out". Most enzymes will come up
slower, and we will stain them as soon as we have them sliced,
and the person scoring will be able to score them in the order
in which they develop. Some enzymes will take from 15 to 30 minutes,
others may take up to an hour or more for a good expression of
that particular loci.
Q. Why do the starch gels
need to be sliced in order to stain?
A. We are able to pull many
viable loci from a gel, up to 6 on some gels. We slice the gel
for many reasons. The first and most important is that in order
to retrieve more than one loci, we need more than one stain. After
staining a gel, the heat of the stain and incubator will often
render any other loci in that slice unreadable. Heat will often
destroy enzymes, so it's a one shot deal with staining. Once a
slice is stained, it rarely will be good for staining any loci,
other than the original one targeted. Also, the top slice of a
gel usually thickens up during the running process, making it
almost impossible to read accurately any loci from that slice.
Q.
What is the purpose of the incubator?
A. Once the gel slice has
been stained for a particular loci, the stain will develop faster
in heat. This is much like developing a picture in that the picture
will come up faster in certain chemicals and low light. Too much
light or chemicals, and the picture will over-develop, too little,
and it will be unscoreable due to under-developing. |
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