Data Sheet - BioVision
Contents
1. 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 For research use only Page 2 of 22. BioVision Alanine Colorimetric Fluorometric Assay Kit Catalog K652 100 100 reactions Store kit at 20 C Introduction Alanine is the 2 most abundant of the 20 proteinogenic amino acids Nonessential it plays a key role in the glucose alanine cycle between tissues and liver In muscle and other tissues that degrade amino acids amino groups are pooled as glutamate by transamination Glutamate then transfers the amino group to pyruvate via alanine aminotransferase forming alanine and a ketoglutarate The alanine is passed into the blood and transported to the liver A reverse of the alanine aminotransferase reaction takes place in liver Pyruvate can be used in gluconeogenesis to form glucose which may return to other tissues through the circulatory system There appears to be a correlation between alanine levels and higher blood pressure energy intake cholesterol levels and body mass index BioVision s Alanine Assay Kit provides a simple sensitive detection method of alanine In the kit alanine is converted to pyruvate which is specifically detected leading to proportional color A 570nm 0 10 nmol or fluorescence Ex Em 535 587nm 0 1 nmol development Serum concentration 24 76 pg ml 3 9 nmol 10 ul Kit Contents Components K652 100 Cap Code Part No Alanine Assay Buffer 25 ml WM K652 100 1 Alanine probe in DMSO 0 2 ml Red K652 100 2A Alanine Converting Enzyme lyophilized Purple K652 100 4 Alanine Development
3. Mix lyophilized Green K652 100 5 Alanine Standard 10 umol lyophilized Yellow K652 100 6 Storage and Handling Store the kit at 20 C protect from light Allow Assay Buffer to warm to room temperature before use Briefly centrifuge vials before opening Read the entire protocol before performing the assay Reagent Reconstitution and General Consideration Alanine Probe Ready to use as supplied Warm to room temperature to melt frozen DMSO prior to use Protect from light and moisture Stable for 2 months at 20 C Alanine Converting Enzyme Development Enzyme Mix Dissolve separately with 220 ul Assay buffer Pipette up and down to dissolve Aliquot into portions and store at 20 C Avoid repeated freeze thaw cycles Use within two months Alanine Standard Dissolve in 100 ul dH2O to generate 100 mM 100 nmol ul Alanine Standard solution Keep cold while in use Store at 20 C Ensure that the Assay Buffer is at room temperature before use Keep the Alanine Enzyme Mix on ice during the assay and protect from light Alanine Assay Protocol 1 Alanine Standard Curve Colorimetric Dilute 10 ul of the 100mM Alanine standard with 990 ul DI H20 to generate 1 mM standard Alanine Add 0 2 4 6 8 10 ul of the diluted Alanine 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 Fluorimetric Dilute standard as for the colorimetric procedure then t
4. ake 100 ul of the 1 mM standard and add to 900 ul DI H20 to make 0 1mM Alanine standard Add 0 2 4 6 8 10 ul of the diluted Alanine standard into a 96 well plate to generate 0 0 2 0 4 0 6 0 8 1 0 nmol well standard Bring the volume to 50 ul with Assay buffer 2 Sample Preparation Tissues or cells 1 x108 can be homogenized in 100 ul Assay Buffer centrifuge to remove insoluble material at 13 000 g 10 minutes 10 50 ul deproteinized serum samples can be directly diluted in the Assay Buffer Bring sample wells to 50 ul well with Assay Buffer in a 96 well plate For unknown samples we suggest testing several doses of your sample to make sure the readings are within the standard curve range BioVision Incorporated 155 S Milpitas Boulevard Milpitas CA 95035 USA rev 2 13 For research use only 3 Reaction Mix Mix enough reagent for the number of assays to be performed For each well prepare a total 50 ul Reaction Mix containing Alanine Measurement Background Control Assay Buffer 44 ul 46 ul Alanine Converting Enzyme 7 o ee Alanine Development Mix 2 ul 2 ul Alanine Probe 2 ul 2 ul Use background control if high levels of pyruvate are suspected to be in the samples For the fluorescent assay dilute the probe 5 10X to reduce background Add 50 ul of the Reaction Mix to each well containing Alanine standard test and background control samples Mix well Incubate the reaction for 60 min at 37 C protect from ligh
5. oVision GENERAL TROUBLESHOOTING GUIDE rev 2 13 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 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 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 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
6. t 4 Measure O D at 570 nm in a microplate reader or fluorescence using Ex Em 535 587 nm 5 Calculation Correct background by subtracting the value derived from the 0 Alanine control from all sample readings The background reading can be significant and must be subtracted from sample readings Plot Alanine standard Curve Alanine concentrations of the test samples can then be calculated C S S nmol ul or mM where S is the sample amount of unknown in nmol from standard curve Sy is sample volume ul added into the wells L Alanine Molecular Weight is 89 1 g mol 1 2 Q 2 6 1 oe 0 8 E4 E S S 06 2 D N 3 y 7142 6x 150 29 a 04 y 0 1232x 0 0203 re 0 2 ac 0 o 0 0 2 0 4 0 6 0 8 1 0 2 4 6 8 10 L Alanine nmol L Alanine nmol RELATED PRODUCTS NAD NADH Quantification Kit ADP ATP Ratio Assay Kit Glucose Assay Kit Ethanol Assay Kit Pyruvate Assay Kit Creatine Assay Kit Ammonia Assay Kit Triglyceride Assay Kit Choline Acetylcholine Quantification Kit Nitric Oxide Assay Kit NADP NADPH Quantification Kit Ascorbic Acid Quantification Kit Fatty Acid Assay Kit Uric Acid Assay Kit Lactate Assay Kit Il L amino Acid Assay Kit Free Glycerol Assay Kit Hemin Assay Kit Total Antioxidant Capacity TAC Assay Kit Glutathione Detection 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 Bi
7. thawed components Use of expired kit or improperly stored reagents Allowing the reagents to sit for extended times on ice e Incorrect incubation times or temperatures e 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 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
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