KIT CONTENTS

Assay Buffer: 12 mL Enzyme A: 120 μL Enzyme B: 120 μL

Dye Reagent: 120 μL Standard: 50 μL 50 mg/mL

Storage conditions. The kit is shipped on ice. Store all reagents at -20°C. Shelf life of six months after receipt.

Precautions: Reagents are for research use only. Normal precautions for laboratory reagents should be exercised while using the reagents. Please refer to Material Safety Data Sheet for detailed information.

Soluble Starch. Grind up 5-10 mg sample, wash off any free glucose and small oligosaccharides with 1 mL 90% ethanol, warm to 60°C for 5 minutes with occasional vortexing. Centrifuge at 10,000g for 2 minutes. Decant the supernatant. Repeat the wash twice. Remove ethanol. Soluble starch in the pellet is extracted with 1 mL H2O incubated in a boiling water bath for 5 minutes. Spin 10,000g for 2 minutes. The supernatant is soluble starch and resistant starch is in the insoluble pellet.

Resistant Starch. After extracting soluble starch, extract the water insoluble pellet with 0.2 mL DMSO and heat in boiling water bath for 5 minutes. Dilute sample 1:100 in H2O prior to assay. Alternatively, resistant starch can be extracted with KOH/H3PO4 or KOH/acetate method [1].

1. Equilibrate all components to room temperature. During experiment, keep thawed enzymes in a refrigerator or on ice.

2. Standards and samples: Dilute standard by mixing 5 μL Standard with 1.25 mL dH2O to give 200 μg/mL standard. Dilute standard in dH2O as follows.

Transfer 10 μL standard and samples into separate wells of a clear flat-bottom microplate.

3. Working Reagent. For each reaction well, mix 90 μL Assay Buffer, 1 μL Enzyme A, 1 μL Enzyme B and 1 μL Dye Reagent in a clean tube. Transfer 90 μL Working Reagent into each reaction well. Tap plate to mix.

4. Incubate 30 min at room temperature. Read optical density at 570nm (550-585nm).

For fluorimetric assays, the linear detection range is 0.2 to 20 μg/mL starch. Follow steps 1-3 of the colorimetric procedure, but prepare 0, 5, 10, 15 and 20 μg/mL Standard and use a black flat-bottom microplate. Incubate 30 min at room temperature and read fluorescence at lex = 530nm and lem = 585nm.

Subtract Blank reading (OD570nm or fluorescence intensity) from the standard reading values and plot the DOD or DF against standard concentrations. Determine the slope and calculate the starch concentration of the sample.

RSAMPLE and RBLANK are the OD570nm or fluorescence intensity values of the sample and blank (water, or sample blank, see below).

1. This assay is based on a kinetic reaction, the use of a multichannel pipettor for adding the working reagent is recommended.

2. Interference. Interference. SH-group containing reagents (e.g., DTT, β-mercaptoethanol) may interfere with this assay and should be avoided in sample preparation.

Pipeting devices, centrifuge tubes, clear flat bottom 96-well plates and plate reader.