I am doing a 4-MUG assay for GUS expression in seeds. I imbibe them for 6 hours and 9 hours (two separate experiments).
Is this a basal level of fluorescence? I used 1 mM 4-MUG. I also measured just 4-MUG and stop solution and subtracted my samples with others.
I only incubated for 30 min.
This is my 4- MU standard curve trend line formula: y = 1024.7x - 764.07, which I normalized to.
I followed this protocol (with some modifications):
Kim KW., Franceschi V.R., Davin L.B., Lewis N.G. (2006) β-Glucuronidase as Reporter Gene. In: Salinas J., Sanchez-Serrano J.J. (eds) Arabidopsis Protocols. Methods in Molecular Biology™, vol 323. Humana Press
Please if anyone has any advice or reasons why my Columbia may be showing GUS expression, please let me know!
Did you mean that your 'non-transgenic' Columbia Arabidopsis plants showed GUS expression?
yes, they are non transgenic and they show no staining however there is noise from the 1 mM 4-MUG I'm guessing, and I ran a blank well and subtracted but i still got some expression, I was just wondering if that's normal or not ?
seeds coat may have high endogenous GUS level.
Try to avoid seeds coat. here is some discussion:Article Factor affecting the endogenous β-glucuronidase activity in ...
It is normal to get some basal level of Gus expression in the control samples. You have to run multiple control samples and fix a value that will be used for normalizing your data. Try running at least 10 replicates of columbia from different leaves and check the average fluoresecene n standard deviation.
Dear Malaika Gomes,
Many plant species show GUS 'background activity'. Please see the paper mentioned below:
"Ubiquitous presence of β-glucuronidase (GUS) in plants and its regulation in some model plants" (2006 Planta)
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