How to assay swelling of membrane vesicles

Daniel Kim dkim at nmsu.edu
Thu Apr 13 17:23:03 EST 1995


Dear Group:

I am trying to start a thesis in which I study the receptor binding 
properties of the insecticidal protein from Bacillus thuringiensis (Bt).  
One assay for Bt toxicity is to measure increases in amino acid 
premeability of midgut brush border membrane vesicles from susceptible 
insects (Bt acts by binding and inserting into the cell membrane of 
midgut epithelial cells, forming pores, and killing the cells by osmotic 
lysis . . . or that is a good-enough description).

An alternative assay for Bt toxicity is to prepare brush border membrane 
vesicles and shrink them by placing them into hyperosmotic solutions.  
After osmotic shrinkage, vesicles that have been permeabilized by Bt 
activity will re-swell as pores are formed.

This change in vesicle volume is measured by 90 degree light scattering. 
A nicer way to do the job compared to measuring leakage of [3H]-amino acids.

My question is:

I don't really have access to equipment for 90 degree light scattering.  
Is there some other way to detect changes in vesicle volume?

Also, it would be nice to be able to physically separate re-swelled 
(re-swollen?) vesicles from those that are not permeabilized.  Is there a 
change in density/sedimentation that I could use to advantage from this 
kind of treatment?

I know these questions are a bit vague. . . I ask because I do not know :)

Daniel Kim
dkim at verdi.nmsu.edu



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