Monday, August 4, 2014

GUS Assay

A lot of scientists use reporter genes to quickly analyze/visualize various different cellular features or functions. A reporter gene will make a protein that is easily measured, a lot of them glow like GFP (green fluorescent protein). One can attach any promoter to the gene, letting scientists control when and/or where the protein is produced allowing reporter systems to be highly specialized.

We use a GUS-GFP reporter in our lab. GFP comes from jellyfish and glows green under special light. Simply look under the fluorescent microscope and if you see glowing green, then your gene is active. Since finding an available fluorescent scope can be hard, I prefer to use the GUS portion of our reporter gene.

GUS is one of the reporter genes that we utilize in some of our transformed plant lines. GUS encodes the protein β-glucuronidase, which breaks down complex carbohydrates. Basically it takes great big "sugars" and breaks them into smaller ones. There are two main "food" sources for GUS, X-Gluc and MUG. X-Gluc will result in a visual blue color. MUG results in a small glowing particle.
My cartoon version of GUS "eating" MUG, he'll spit out MU which will glow!

I utilize MUG which will break down into the fluorescent MU. First, I have to measure the amount of protein in my sample so that I can load the same amount of protein to each sample. This normalization step is critical. Since we are looking at the amount of GUS enzyme activity, we need to be sure that we started with a standard amount of protein. That way, any changes that we not are from the enzyme's activity, not extra protein in well A1 vs A2, etc.
Once the protein levels are measured, a standard amount is loaded into each wells of a black plate. Then I add a set amount of MUG. This step is also critical, if one well has more or less MUG "food" then the difference in GUS enzyme activity could be because there was more "food" not more GUS activity. 
Be careful to avoid the molecular biologists arch nemesis - pipette errors!
Next I take this plate and put it into a spectrofluorometer, which can measure the amount of fluorescence in each well. The faster GUS breaks MUG down, the faster the fluorescence will rise. We can use the slope of the resulting line to determine the GUS activity level.
Spectrofluorometer screen shot

Remember, the gene we introduced is the only way that GUS can be present in our samples. The promoter we choose to run the gene must activate it. If the gene is highly up-regulated, lots of GUS will be made and you will see a steep slope (for example B6 in plate above). If the gene is moderately up-regulated, less GUS will be made (like A6). If it is not produced, you will get no change in the slope.

A GUS assay takes about an hour and half. Thanks to a dear friend of mine being able to teach me some streamlining tricks, the processing of that data into useable information takes about an hour.  It's a very powerful, and efficient assay to examine how our promoter responds to various conditions.

1 comment:

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