Question

Trouble with 1,9-dimethylmethylene blue (DMMB) assay to determine sGAG

I have been trying a DMMB assay to determine the sGAG from alginate beads. Still I haven't used any cells in the beads. I just make beads mixed with Chondroitin sulfate solution to standardize. First I made my standard curve using only chondroitin sulfate solution of different dilution but the problem is when I tried with alginate beads, the colour of DMMB dye is not changed (it should be changed from blue to purple). I used DMMB dye with pH 3.00 but in some papers I found it should be 1.5 for alginate beads but at pH 1.5 the color remains the same after mixing the sample with dye. On the other hand, at pH 3.00, the result cannot be accepted. I follow all the required protocols for bead preparation, bead dissolving, dye preparation but i could not get the result. Can anyone advise what I should do?

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All Answers (1)

  • Chunhua Zheng · Stanford University
    Hi Maruf,

    Are you getting signal saturation at pH 1.5? What are you using to create your standard curve? Is your standard curve linear? If you are getting saturation, then you should dilute your sample until the reading falls within the linear range of your standard curve.

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