My GUS staining solution contained phosphate buffer, ferricyanide, ferrocyanide, triton X-100, EDTA, methanol and X-Gluc. Except for X-Gluc, anyone know the function or importance of other components in that solution?
From the attached paper (Texas A & M University [a]), the GUS staining solution is:
"X-gluc substrate solution: Dissolve 1 mg 5-bromo-4-chloro-3-indolyl -D-Glucuronide (X-Gluc) in 0.1 mL methanol (see Note 3), add 1 mL 2x buffer (see Note 4), 20 μL 0.1 M potassium ferrocyanide, 20 μL 0.1 M potassium ferricyanide (see Note 5), 10 μL 10 % (w/v) solution of Triton X-100, 0.85 mL. water."
(1) X-gluc: the substance for gus gene encodes enzyme (glucuronidase) to work on.
(2) Methanol: used to dissolve X-gluc
(3) buffer: with right pH for the enzyme to work in
(4) potassium ferrocyanide and potassium ferricyanide: The initial product of glucuronidase hydrolysis of X-Gluc has no color. Instead, the initial indoxyl derivative produced must undergo an oxidative dimerization to form the insoluble and highly colored indigo dye. This dimerization is stimulated by atmospheric oxygen, and can be enhanced by using an oxidation catalyst such as a K+ ferricyanide/ferrocyanide mixture. [ b ]
(5) Triton X-100: used as a surfactant [ c ]
[a] https://microscopy.tamu.edu/wp-content/uploads/2018/08/GUS_Localization_in_plants.pdf
[b] https://www.goldbio.com/documents/1053/Gus%20Gene%20Assay%20Protocol.pdf
[c] Article Development of Agrobacterium-mediated transient expression s...
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