DMMB assay for sulphated proteoglycans

100mM K2HPO4 pH 8.0

make up:

0.5l 1M K2HPO4 (87.09g in 500ml dH2O)

0.5l 1M KH2PO4 (68.045g in 500ml dH2O)

mix: 94ml of K2HPO4 and 6ml of KH2PO4, top up to 900ml, pH to 8.0 using KH2PO4

0.016g DMMB

3.04g glycine

2.37g NaCl

9.5ml 0.1M HCl

top up to 1l with dH2O

note: the final solution should be pH 3.0 and have absorbance of 0.31 at λ=525

store in a dark bottle away from light

make up chondroitin sulphate standard dilutions starting at 50ug/ml to 0.05ug/ml in the K2HPO4 buffer

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use 5ul sample per well of the 96-well plate, plate samples in duplicate

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add 125ul of DMMB soution using a multichannel pipette

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read absorbance immediately, at λ=525

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normalise the readout to the BCA assay (or a DNA measurement in case of tissue digests)