Data Sheet - BioVision
Contents
1. AD P H Quantification Kit Ascorbic Acid Quantification Kit Total Antioxidant Capacity TAC Assay Kit Ethanol Assay Kit Pyruvate Assay Kit Creatinine Assay Kit Ammonia Assay Kit Triglyceride Assay Kit Alanine Assay Kit Sarcosine Assay Kit Phenylalanine Assay Kit ADP ATP Ratio Assay Kit Glutathione Detection Kit Fatty Acid Assay Kit Uric Acid Assay Kit Lactate Assay Kit amp Il Nitric Oxide Assay Kit Free Glycerol Assay Kit Hemin Assay Kit Glucose Assay Kit L Amino Acid Assay Kit Cholesterol Assay Kit 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 GENERAL TROUBLESHOOTING GUIDE rev 02 13 For research use only Problems Cause Solution Assay not working e Use of ice cold assay buffer e Omission of a step in the protocol e Plate read at incorrect wavelength e Use of a different 96 well plate e Assay 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 Luminescence White plates Colorimeters Clear plates Samples with erratic readings e Use of an incompatible sample type e Samples prepared in a different buffer e Samples were not deproteinized if indicated in datasheet Cell tissue samples were not completely homogenized e Samples use2. BioVision Branched Chain Amino Acid Leu Ile Val Colorimetric Assay Kit Catalog K564 100 100 Reactions Store kit at 20 C Introduction The branched chain amino acids or BCAA s refer to the amino acids with non linear aliphatic side chains namely leucine isoleucine and valine These three essential amino acids make up approximately 1 3 of skeletal muscle in the human body BCAA s are currently used clinically to aid in the recovery of burn victims as well as for strength supplementation for athletes BCAA s primarily Leu can stimulate insulin secretion The BCAA s have also been implicated in a wide range of other physiological effects BioVision s BCAA Assay Kit provides a simple convenient means of measuring the BCAA s in a variety of biological samples The kit utilizes an enzyme assay in which BCAA is oxidatively deaminated producing NADH which reduces the probe generating a colored product Amax 450 nm BioVision s BCAA kit measures BCAA s in the range of 0 to 10 nmol per sample with a detection limit of 0 2 nmol 10 uM BCAA in sample BCAA s are present in serum 0 1 0 4 mM each 0 125 1 5 mM combined Kit Contents Components K564 100 Cap Code Part No BCAA Assay Buffer 25 ml WM K564 100 1 BCAA Enzyme Mix lyophilized Green K564 100 2 WST Substrate Mix lyophilized Red K564 100 3 Leu Standard 1 umol 100 ul Yellow K564 100 4 Storage and Handling Store the k
3. d after 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 Use the assay buffer provided in the kit or 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 Aliquot and freeze samples if needed to use multiple times Troubleshoot if needed deproteinize samples Use fresh samples or store at correct temperatures till use Lower Higher readings in Samples and Standards e Improperly thawed components e Use of expired kit or improperly stored reagents e Allowing the reagents to sit for extended times on ice e Incorrect incubation times or temperatures e Incorrect volumes used Thaw all components completely and mix gently before use e Always check the expiry date and store the components appropriately 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 e Pipetting errors in the standard e Pipetting errors in the reaction mix e Air bubbles formed in well Standard stock is at an incorrect concentration e Calculat
4. er of assays to be performed For each well prepare a total 50 ul Reaction Mix containing Amino Acid Measurement 3 Reaction Mix Bkgd Control Assay Buffer 46 ul 48 ul Enzyme Mix Q2ub 0 wae WST Substrate Mix 2 ul 2 ul Add 50 ul of the Reaction Mix to each well containing the leucine standard and test samples Mix well Incubate the reaction for 30 min at room temperature protect from light NADH and NADPH can generate significant background If these compounds are suspected of being in your sample at significant concentration perform a simple background control by replacing the Enzyme Mix with 2 ul Assay Buffer The background reading should be subtracted from the BCAA test sample readings Measure O D at 450 nm in a microplate reader Calculation Correct background by subtracting the value derived from the 0 BCAA standards from all readings The background reading can be significant and must be subtracted from sample readings Plot standard curve Apply sample readings to the standard curve BCAA concentrations of the test samples can then be calculated C S S nmol ul or mM Where Sa BCAA content of unknown samples nmol from standard curve S sample volume ul added into the assay wells BCAA molecular weights are Leu 131 18 lle 131 18 Val 117 15 g mol 0 7 y 0 0684x 0 0064 2 4 6 BCAA nmol well 8 to Leucine Assay performed according to this protocol RELATED PRODUCTS NAD P N
5. ion errors e Substituting reagents from older kits lots Thaw and resuspend all components before preparing the reaction mix e Avoid pipetting small volumes Prepare a master reaction mix whenever possible 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 e Samples contain interfering substances e Use of incompatible sample type e 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 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 2
6. it at 20 C protect from light Allow Assay Buffer to warm to room temperature before use Briefly centrifuge vials prior to opening Read the entire protocol before performing the assay Reagent Reconstitution and General Consideration BCAA Enzyme Mix Dissolve with 220 yl BCAA Assay Buffer Pipette up and down to dissolve Stable at 4 C for two months WST Substrate Mix Dissolve with 220 ul of dH2O before use Mix well store at 4 C protect from light Stable for 2 months Leucine Standard Ready to use as supplied Store at 4 C BCAA Assay Protocol Standard Curve Dilute 10 ul of the 10 mM Leucine Standard with 90 ul dH2O to generate 1 mM Leucine standard Add 0 2 4 6 8 10 ul of the diluted Standard into a 96 well plate to generate 0 2 4 6 8 10 nmol well standard Bring the volume to 50 ul with Assay Buffer Sample Preparation Tissue 20 mg or cells 2 x 10 can be homogenized with 100 ul Assay buffer Centrifuge at 15 000g for 10 minutes to remove cell debris and other insoluble materials Add samples to sample wells in a 96 well plate and bring the volume to 50 ul well with Assay Buffer We suggest testing several doses of your sample to make sure the readings are within the standard curve range Typical volume for serum samples should be in the range of 1 20 ul BioVision Incorporated 155 S Milpitas Boulevard Milpitas CA 95035 USA rev 02 13 For research use only Mix enough reagents for the numb
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