Starch, chemical formula (C6H10O5), is a polysaccharide carbohydrate consisting of a large number of glucose units joined together by glycosidic bonds. All plant seeds and tubers contain starch present in the form of amylose and amylopectin. Starch is the most consumed polysaccharide in the human diet. Some starches are digested very quickly, and cause a rapid and large rise in blood sugar. Others are digested more slowly, and some starch, called resistant starch, is not digested in the small intestine at all, and thus causes little or no blood sugar rise.
Simple, direct and automation-ready procedures for measuring starch concentrations find wide applications in research and drug discovery. Starch Assay Kit uses a single Working Reagent that combines the enzymatic break down of starch and the detection of glucose in one step. The color intensity of the reaction product at 570nm or fluorescence intensity at lem/ex=585/530nm is directly proportional to the starch concentration in the sample. This simple convenient assay is carried out at room temperature and takes only 30 min.
Key Features:Use as little as 10ul samples. Linear detection range: 2 to 200 ug/mL starch for Colorimetric Assay:s and 0.2 to 20 ug/mL for Fluorometric Assay:s.
Kit Contents:Assay Buffer: 12ml Enzyme A: 120ul Enzyme B: 120ulDye Reagent: 120ul Standard: 50ul 50 mg/mLStorage 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.
Sample Preparation:Soluble Starch. Grind up 5-10 mg sample, wash off any free glucose and small oligosaccharides with 1ml 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 1ml 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 0.2ml 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].
Colorimetric Procedure: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 5ul Standard with 1.245ml dH2O to give 200 ug/mL standard. Dilute standard in dH2O as follows
No 200 ug/mL STD+H2O Vol (ul) Starch (ug/ml)1 200ul+0ul 200 2002 150ul+50ul 200 1503 100ul+100ul 200 1004 50ul+150ul 200 505 0ul+200ul 200 0Transfer 10ul standard and samples into separate wells of a clear flat-bottom microplate
3. Working Reagent. For each reaction well, mix 90ul Assay Buffer, 1ul Enzyme A, 1ul Enzyme B and 1ul Dye Reagent in a clean tube. Transfer 90ul Working Reagent into each reaction well. Tap plate to mix
4. Incubate 30 min at room temperature. Read optical density at 570nm (550-585nm).
Fluorometric Procedure:For Fluorometric Assays, the linear detection range is 0.2 to 20 ug/mL starch. Follow steps 1-3 of the Colorimetric Procedure:, but prepare 0, 5, 10, 15 and 20 ug/mL Standard and use a black flat-bottom microplate. Incubate 30 min at room temperature and read fluorescence at lex=530nm and lem=585nm.
Calculation: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
Starch =RSAMPLE-RBLANKSlopeug/mLRSAMPLE and RBLANK are the OD570nm or fluorescence intensity values of the sample and blank (water, or sample blank, see below).
General Considerations: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, b-mercaptoethanol) may interfere with this assay and should be avoided in sample preparation.
Materials Required, But Not Provided:Pipeting devices, centrifuge tubes, clear flat bottom 96-well plates and plate reader.
