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Purple Membrane Lab

THE PROCEDURE:

This lab was guided by Marc Facciotti to help the interns get a better understanding of what the scientist do in the lab. They wanted to isolate bacteriorhodopsin from halobacterium. To isolate this protein water was added to the cell, which caused the salinity level to drop inside the cell and in result the cell exploded.

After the explosion, the membrane fractions were by centrafugetation collected. Bacteriorhodopsin likes to stay together so, they collected the membrane pieces that were bigger.

Subsequent[ly] to the collection the interns created a solution containing four different concentrations of sucrose and sodium azide. Sodium azide is a poison that was used in this experiment to kill any bacteria that may grow and eat Bacteriorhodopsin.

Then the interns created a gradient with this solution of: 30%, 40%, 50%, and 60% of the sucrose and sodium azide solution. Marc told the interns that the protein, Rhodopsin should end up around the 40-50 percent of the gradient. The students put the Halobacterium on the top of the gradient and put it in the ultracentrifuge for two hours.

When they took it out they found that the two places where the membrane ended up at where 50% and 60%. After that they took the membrane out and took the cell density by using the spectrophotometer and was then ran in a gel to determine the small strands from the big strands and to get a better understanding about how the protein behaves, how it looks like, how big it is and where it is located.

 

 

 

 

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Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation.