DMMB assay for sGAG determination - (Jul/26/2012 )
I have been trying 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 same after mixing the sample with dye. on the other hand, at pH 3.00, the result can not be accepted. I follow all the required protocols for bead preparation, bead dissolving, dye preparation. But i could not get the result. Now what should I do? I feel very helpless.
hello,may be your problem is one bead contains too little GAG to test,try 3 beads.