vacuole transport lab?

Ross Koning Koning at ECSUC.CTSTATEU.EDU
Thu Nov 7 08:13:50 EST 1996


At  1:19 PM 11/6/96 -0800, HELM at siena.edu wrote:
>Hi folks,  Does anyone out there know of a good lab to illustrate transport
>processes in vacuoles, or other membranes for that matter.  I've seen some
>intuiging stuff dealing with beat vacuoles, but don't have a fluorometer with
>which to measure acridine quenching. I'd really like to put together a lab
>demonstrating these concepts (sucrose/H+ antiporter and the like), but haven't
>yet located some good, reliable protocols.  Thanks much, Ken
>

Ken,

You probably know about the old beet root cylinder
project.  You cut cylinders with a cork borer, cut
to length with a razorblace, rinse with distilled
water until the rinse is colorless, then place
cylinders in various solutions of a standard volume,
incubate for a standard length of time.  At the end
of the elapsed time, you decant the liquid to
spectrophotometer tubes/cuvettes and observe absorbance
at 540 nm (or about).

The solutions of some interest include:

a strong sodium chloride solution
a weak calcium chloride solution
a combination of these
a distilled water control.

an acetone solution

a freezing pre-treatment
a boiling pre-treatment

etc. as you wish.

The betacyanin is located in the vacuole and
leaks out under some of these situations.

I would expect if you are doing something with
isolated vacuoles, that you could use those
instead of whole-tissue cylinders.  I do not
know of any reason why vacuoles from beet root
could not be isolated (though that might exist!).

I like the exercise because it teaches about
membrane stability, frost tolerance, and gives
an example about the effects of denaturation of
membrane proteins so students realize that they
exist for a reason.  Fresh beet roots are available
in our grocery stores routinely.  The only caution
is that sometimes purchased roots get frozen by a
malfunctioning refrigerator or in transit to the
store in the winter.  In winter I usually buy from
two different stores so that if one set is no good
the other might be OK.  Any extras go home for dinner.
(I have to buy supplies like this out-of-pocket for
my classroom because I teach at an underfunded state
school).

This exercise might not address the central issue
about the tonoplast that you wanted, but maybe it
gives you some ideas about how you would alter it
to reach your objectives...and then share your
results with the list.

Good Luck!

ross

______________________________________________________________
                            |
Ross Koning                 | Koning at ecsu.ctstateu.edu
Biology Department          | http://koning.ecsu.ctstateu.edu/
Eastern CT State University | Phone: 860-465-5327
Willimantic, CT  06226  USA | Fax: 860-465-5213
____________________________|_________________________________

                Plant Physiology is Phun!

 /\|___/\     //\______COOH   NH-CH2-CH=C-CH2OH  \/OH
|  |  |  |    |  |  ||       //\___     \CH3     /\|/\\/\\COOH
 \/ \/|\/|    \\/ \ /       N  ||  N            |  |
 /\ | |__|=        NH       |  || ||           //\//\
  | COOH                    \\ /\ /            O
  COOH        H2C=CH2         N  NH
______________________________________________________________





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