Proteasome Function In Mammalian Cells

November 22, 2016

Colorado State University
Food Science and Human Nutrition
Graduate Student

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Quality of Results

Ease-of-Optimization

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Company:

Clontech, Takara

Product Name:

Proteasome Sensor Vector

Catalog Number:

632425

The goal of this experiment was to determine the activity level of the proteasome in H4IIE rat hepatoma cells due to a variety of treatments. This was chosen because we could easily view activity under a microscope.

Experimental Design and Results Summary

Application

Fluorescent Microscopy

Starting Material

Fixed H4IIE rat hepatoma cells

Protocol Overview

Using a combination of lipofectamine with the plus reagent from thermo scientific (plasmic transfection reagent), we transfect 0.5ug/uL of DNA into the rat heptoma cells. After 4.5 hours, the cells are trypsinized and split. Then we treat our cells using our normal treatment conditions

Tips

Transfection efficiency is low. Try different amounts of DNA and different transfection times.

Results Summary

Using a proteasome inhibitor such as MG132 as a positive control, this experiment turned out to work pretty well. There is a clear increase in GFP fluorescence when the proteasome inhibitor is present which is what we were looking for.

DOI or PMID #

N/A

Additional Notes

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Summary

The Good

This seems to work with our basic transfection protocol, although it took a lot of tweaking.

The Bad

Hard to optimize. Some times just doesn't work at all and we don't know why.

The Bottom Line

This vector is an interesting way to go about looking at proteosome activity. The GFP is fused to a degradation domain, targeting the GFP for rapid degradation. Therefore when the proteasome is not inhibited, this protein will NOT accumulate in cells, be degraded and there will be essentially nothing to view under the microscope and no fluorescence.However, if the proteasome is inhibited, this protein builds up within the cells and causes an increase in GFP fluorescence.It's an odd way to go about it, but using a proteasome inhibitor such as MG132 as a positive control, we have had some success.

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