The glucose and mannose standard (bottle 8) is just a standard solution of these sugars, there is no glucomannan present so glucomannan calculation and hydrolysis is not required for this standard.
0.1 mL of the standard should be applied to the assay.
Please see as follows:
Pipette into cuvettes | Blank | Sample | Standard |
distilled water (at ~ 35°C) sample solution suspension 5 (β-Gos/β-Mos) | 0.02 mL 0.50 mL - | - 0.50 mL 0.02 mL | 0.42 mL |
Mix*, then cap the cuvettes and incubate at 35°C for 20 min. Then add: | |||
distilled water (at ~ 35°C) solution 2 (buffer) solution 3 (NADP+/ATP) | 2.00 mL 0.20 mL 0.10 mL | 2.00 mL 0.20 mL 0.10 mL | 2.00 mL 0.20 mL 0.10 mL |
Mix**, incubate at ~ 25-30°C, read absorbances of the solutions after approx. 3 min (A1) and start the reactions by addition of: | |||
suspension 6 (HK/G6P-DH) | 0.02 mL | 0.02 mL | 0.02 mL |
Mix** and read the absorbances of the solutions (A2) at the end of the reaction (approx. 5 min). If the reaction has not stopped after 5 min, continue to read the absorbances at 2 min intervals until the absorbances remain the same over 2 min. Then add: | |||
suspension 7 (PGI/PMI) | 0.02 mL | 0.02 mL | 0.02 mL |
Mix** and read the absorbances of the solutions (A3) at the end of the reaction (approx. 20 min). If the reaction has not stopped after 20 min, continue to read the absorbances at 2 min intervals until the absorbances remain the same over 2 min. | |||
The glucose / mannose content in the standard should be confirmed as follows:
For glucose this equates to:
((2.84 x 180.16) / (6300 x 1 x sample volume)) X ΔA glucose
For mannose this equates to:
((2.86 x 180.16) / (6300 x 1 x sample volume)) X ΔA mannose