Data Sheet - BioVision
Contents
1. lots e Thaw and resuspend all components before preparing the reaction mix e Avoid pipetting small volumes e Prepare a master reaction mix whenever possible e Pipette gently against the wall of the tubes e Always refer the dilutions in the data sheet e Recheck calculations after referring the data sheet e Use fresh components from the same kit Unanticipated results e Measured at incorrect wavelength Samples contain interfering substances e Use of incompatible sample type Sample readings above below the linear range e Check the equipment and the filter setting e Troubleshoot if it interferes with the kit e Refer data sheet to check if sample is compatible with the kit or optimization is needed e Concentrate Dilute sample so as to be in the linear range Note The most probable list of causes is under each problem section Causes Solutions may overlap with other problems BioVision Incorporated 155 S Milpitas Boulevard Milpitas CA 95035 USA Tel 408 493 1800 Fax 408 493 1801 www biovision com tech biovision com Page 2 of 22. Hydrolysis Enzyme Mix Development Enzyme Mix Dissolve with 220 ul Hydrolysis Buffer Vortex gently to dissolve Keep on ice Store at 20 C Stable for at least two months Starch Assay Protocol Standard Curve Preparations Colorimetric Dilute Starch Standard to 0 2 mg ml by adding 10 ul of the Standard to 90 ul of distilled water mix well Add 0 2 4 6 8 10uI into a series of wells Adjust volume to 50 ul well with Hydrolysis Buffer to generate 0 0 4 0 8 1 2 1 6 and 2 0 ug well of the Starch Standard Fluorometric Dilute Starch Standard to 0 02 mg ml by adding 10 ul of the Standard to 990 ul of distilled water mix well Add 0 2 4 6 8 10 ul into a series of wells Adjust volume to 50 ul well with Hydrolysis Buffer to generate 0 0 04 0 08 0 12 0 16 and 0 2 ug well of Starch Standard Sample Preparation Depending on your assay purpose quantitation mw distribution compartmentalization etc prepare starch samples according to established protocols A Soluble Starch Extraction 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 Soluble starch can be extracted with 1 ml H20 and heating on a boiling water bath for 5 minutes Spin at 10 000g for 2 minutes to remove insoluble materials The supernatant is soluble starch B Re
3. BioVision Starch Colorimetric Fluorometric Assay Kit Catalog K647 100 100 assays Store at 20 C Introduction Starch is a complex carbohydrate consisting of a large number of glucose units All plants contain starch present as amylose linear a 1 4 linked polymer and amylopectin highly a 1 6 branched a 1 4 polymer Starch generally contains 0 25 amylose and 75 100 amylopectin The BioVision Starch Assay Kit provides an easy convenient method to measure starch levels in a variety of samples In the assay starch is hydrolyzed to glucose which is oxidized to generate color Amax 570 nm and fluorescence Ex Em 535 587 nm The assay can detect starch at 0 0004 to 2 mg ml Kit Contents Components K647 100 Cap Code Part Number Hydrolysis Buffer 25ml NM K647 100 1 Development Buffer 25 ml WM K647 100 2 OxiRed Probe 0 4 ml Red K647 100 3A Hydrolysis Enzyme Mix Lyophilized Blue K647 100 5 Development Enzyme Mix Lyophilized Green K647 100 6 Starch Standard 2 0 mg ml 100 ul Yellow K647 100 7 Storage and Handling Store kit at 20 C protect from light and moisture Warm Buffers to room temperature before use Briefly centrifuge all small vials prior to opening Read entire protocol before the assay Reagent Preparation and Storage Conditions OxiRed Probe Ready to use as supplied Warm up gt 18 C to melt frozen DMSO before use Mix well store at 20 C protect from light and moisture
4. l 48 7 ul Development Enzyme Mix 2 ul 1 0 ul OxiRed Probe 2 ul 0 3 ul Add 50 ul of Development Mix to each well containing Starch Standard or samples 5 Incubate at room temperature for 30 minutes protect from light 6 Measure colorimetrically OD 570 nm or fluorometrically Ex Em 535 587 nm 7 Calculation Correct background by subtracting the value of the O starch control from all sample readings Note The background can be significant and must be subtracted Plot standard curve ug well vs OD Apply sample readings to the standard curve to get the amount of starch in the sample wells The starch concentration in the test samples C Ay Sv ug l or mg ml Ay is the amount of starch ug in your sample from the standard curve Sv is the sample volume ul added to the sample well Multiply by the dilution factors Starch molecular size 60 000 glucose molecules MW 10 10 daltons Where glucose y 0 651x 0 027 mcorn y 0 700x 0 006 Apotato y 0 648x 0 012 erice y 0 653x 0 003 y 0 690x 0 002 x wheat 0 0 4 0 8 1 2 1 6 2 Starch yg well Figure 1 Starch Standard Curve Different types of pure starch were extracted with 10N KOH H3PO as described following the kit protocol VII References 1 Quantitative Isolation and Dispersion of Starch from Corn Kernels without Degradation James P McGuire Stig R Erlander Starch Starke Vol 18 No 11 2006 342 346 2 Overview of Labora
5. r multiple free thaw cycles e Presence of interfering substance in the sample e Use of old or inappropriately stored samples e Refer data sheet for details about incompatible samples e Refer data sheet for instructions e Use the 10 kDa spin cut off filter or PCA precipitation as indicated e Use Dounce homogenizer increase the number of strokes observe for lysis under microscope e Aliquot and freeze samples if needed to use multiple times e Troubleshoot if needed deproteinize samples e Use fresh samples or store at correct temperatures till use Lower Higher readings in Samples and Standards Improperly thawed components e Use of expired kit or improperly stored reagents Allowing the reagents to sit for extended times on ice e Incorrect incubation times or temperatures Incorrect volumes used e Thaw all components completely and mix gently before use e Always check the expiry date and store the components appropriately e Always thaw and prepare fresh reaction mix before use e Refer datasheet amp verify correct incubation times and temperatures e Use calibrated pipettes and aliquot correctly Readings do not follow a linear pattern for Standard curve e Use of partially thawed components Pipetting errors in the standard Pipetting errors in the reaction mix Air bubbles formed in well Standard stock is at an incorrect concentration e Calculation errors e Substituting reagents from older kits
6. sistant Starch Extraction After extracting soluble starch extract the water insoluble pellet with 1 ml 10N KOH heat on boiling water bath for 5 minutes Neutralize with 1 ml 10M HPO slowly Spin at 10 000g for 2 minutes to remove insoluble materials The supernatant is resistant starch C Total Starch Extraction After the 90 ethanol wash Step A extract the washed sample directly with 10N KOH H3PO as per the procedure for resistant starch B The supernatant is total starch For starch sample testing Take 20 ul of the extracted starch add 180 ul of Hydrolysis Buffer mix Add up to 50 ul of the diluted sample or buffer blank to test wells Adjust the volume to 50 ul with Hydrolysis Buffer For unknown samples we suggest testing several doses of the sample to ensure the readings are within the standard curve BioVision Incorporated 155 S Milpitas Boulevard Milpitas CA 95035 USA rev 02 13 For research use only 3 Hydrolysis Colorimetric Fluorometric Hydrolysis Enzyme Mix 2 ul 1 ul Mix well incubate for at least 30 minutes at room temperature to hydrolyze starch Note Glucose generates background Glucose control is done without the hydrolysis enzyme add equal volume of Buffer Glucose background can be subtracted from sample reading 4 Development Mix enough reagents for the number of samples and standards For each well prepare a total 50 ul Reaction Mix Colorimetric Fluorometric Development Buffer 46 u
7. tory Isolation of Starch from Plant Materials Thava Vasanthan Current Protocols in Food Analytical Chemistry UNIT E2 1 2001 John Wiley amp Sons Inc 3 A rapid micro starch quantitation method for potato callus and its application with potato tubers J L Varns and J R Sowokinos Journal American Journal of Potato Research Vol 51 No 12 1974 4 Critical study of a procedure for the assay of starch in ligneous plants Gomez L Rubio E Lescourret F Journal of the Science of Food and Agriculture vol 83 no 11 2003 1114 1123 FOR RESEARCH USE ONLY Not to be used on humans Tel 408 493 1800 Fax 408 493 1801 www biovision com tech biovision com Page 1 of 2 BioVision rev 02 13 GENERAL TROUBLESHOOTING GUIDE Problems Cause Solution Assay not working e Use of ice cold buffer Omission of a step in the protocol e Plate read at incorrect wavelength e Use of a different 96 well plate e Buffer must be at room temperature e Refer and follow the data sheet precisely e Check the wavelength in the data sheet and the filter settings of the instrument e Fluorescence Black plates clear bottoms Luminescence White plates Colorimeters Clear plates Samples with erratic readings e Use of an incompatible sample type Samples prepared in a different buffer Samples were not deproteinized if indicated in datasheet Cell tissue samples were not completely homogenized Samples used afte
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