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1. 0 umol Storage and Handling Store kit at 20 C protect from light Warm Citrate Assay Buffer to room temperature before use Briefly centrifuge all small vials prior to opening Reagent Preparation and Storage Conditions Citrate Probe Ready to use as supplied Warm to 37 C for 1 2 min to completely melt the DMSO solution before use Store at 20 C protect from light Use within two months Citrate Developer Enzyme Mix Dissolve with 220 ul Assay Buffer separately Pipette up and down to dissolve Aliquot into portions store at 20 C Avoid repeated freeze thaw cycles Use within 2 months Citrate Standard Dissolve in 100 ul dH20 to generate 100 mM 100 nmol ul Citrate Standard solution Keep on ice while in use Store at 20 C Assay Protocol Standard Curve Preparations Colorimetric Assay Dilute the Citrate Standard to 1 nmol ul by adding 10 ul of the Standard to 990 ul of dH20 mix well Add 0 2 4 6 8 10 ul into a series of standards wells on a 96 well plate Adjust volume to 50 ul well with Assay Buffer to generate 0 2 4 6 8 10 nmol well of the Standard Fluorometric Assay Dilute the Citrate standard to 0 1 nmol ul by adding 10 ul of the standard to 990 ul of dH2O mix well then further dilute by adding 10 ul to 90 ul of dH2O0 Add O 2 4 6 8 10 ul into a series of standards wells on a 96 well plate Adjust the volume to 50 ul well to generate 0 0 2 0 4 0 6 0 8 1 0 nmol well Sample Pr2. BioVision Citrate Colorimetric Fluorometric Assay Kit Catalog K655 100 100 assays Store Kit at 20 C Introduction Citric acid HOOC CH 2 C OH COOH CH2 COOH is a key intermediate in the TCA cycle which occurs in mitochondria It is formed by the addition of oxaloacetate to the acetyl group of acetyl CoA derived from the glycolytic pathway Citrate can be transported out of mitochondria and converted back to acetyl CoA for fatty acid synthesis Citrate is an allosteric modulator of both fatty acid synthesis acetyl CoA carboxylase and glycolysis phosphofructokinase Citrate is widely used industrially in foods beverages and pharmaceuticals Citrate metabolism and disposition can vary widely due to sex age and a variety of other factors BioVision s Citrate Assay Kit provides a simple sensitive and rapid means of quantifying citrate in a variety of samples In the assay citrate is converted to pyruvate via oxaloacetate The pyruvate is quantified by converting a nearly colorless probe to an intensely colored Amax 570 nm and fluorescent E Em 535 587 nm product The Citrate Assay Kit can detect 0 1 to 10 nmoles 2 UM 10 mM of citrate in a variety of samples Kit Contents K655 100 Cap Code Part Number Citrate Assay Buffer 25 ml K655 100 1 Citrate Probe 0 2 ml a K655 100 2 lyophilized Purple K655 100 3 lyophilized Green K655 100 4 lyophilized Yellow K655 100 5 Citrate Enzyme Mix Citrate Developer Citrate Standard 1
3. ERAL TROUBLESHOOTING GUIDE Assay not working e Use of ice cold assay buffer e Assay buffer must be at room temperature e Omission of a step in the protocol e Refer and follow the data sheet precisely e Plate read at incorrect wavelength 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 e Use of a different 96 well plate Clear plates Samples with erratic readings e Use of an incompatible sample type e Refer data sheet for details about incompatible samples e Samples prepared in a different buffer e Use the assay buffer provided in the kit or refer data sheet for instructions e Samples were not deproteinized if indicated in datasheet 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 e Cell tissue samples were not completely homogenized microscope e Samples used after multiple free thaw cycles e Aliquot and freeze samples if needed to use multiple times e Presence of interfering substance in the sample e Troubleshoot if needed deproteinize samples e Use of old or inappropriately stored samples e Use fresh samples or store at correct temperatures till use Lower Higher readings in Samples and Standards e Improperly thawed components e Thaw all components completely and mix gently before use e Use of expired
4. eparation Tissue 20 mg or cells 2 x 10 should be rapidly homogenized with 100 ul of Citrate Assay Buffer Centrifuge at 15 000 g for 10 min to remove cell debris Enzymes in samples may interfere with the assay We suggest deproteinizing samples using a perchloric acid KOH protocol BioVision Cat K808 200 or 10 kDa molecular weight cut off spin columns BioVision Cat 1997 25 Add 1 50 ul sample into duplicate wells of a 96 well plate and bring volume to 50 ul with Assay Buffer We suggest testing several doses of your samples to ensure readings are within the standard curve range BioVision Research Products 155 South Milpitas Boulevard Milpitas CA 95035 rev 07 13 Vi For research use only Develop Mix enough reagent for the number of samples and standards to be performed For each well prepare a total 50 ul Reaction Mix containing Colorimetric Assay Sample Bkgd Control Fluorometric Assay Sample Bkgd Control Citrate Assay Buffer 44 ul 46 ul 44 ul 46 ul Citrate Enzyme Mix 2 ul 2 ul Developer 2 ul 2 ul 2 ul 2 ul Citrate Probe 2 ul 2 ul 2 ul 2 ul Samples may contain oxaloacetate or pyruvate which can generate a background and need to be subtracted from the sample background signal For the fluorometric assay dilute 10X with DMSO to reduce fluorescent background Add 50 ul of the Reaction Mix to each well containing the Citrate Standard and test samples Add 50 ul of the sample background cont
5. kit or improperly stored reagents e Always check the expiry date and store the components appropriately e Allowing the reagents to sit for extended times on ice e Always thaw and prepare fresh reaction mix before use e Incorrect incubation times or temperatures e Refer datasheet amp verify correct incubation times and temperatures e Incorrect volumes used e Use calibrated pipettes and aliquot correctly Readings do not follow a linear pattern for Standard curve e Use of partially thawed components e Thaw and resuspend all components before preparing the reaction mix e Pipetting errors in the standard e Avoid pipetting small volumes e Pipetting errors in the reaction mix e Prepare a master reaction mix whenever possible e Air bubbles formed in well e Pipette gently against the wall of the tubes e Standard stock is at an incorrect concentration e Always refer the dilutions in the data sheet e Calculation errors e Recheck calculations after referring the data sheet e Substituting reagents from older kits lots e Use fresh components from the same kit Unanticipated results e Measured at incorrect wavelength e Check the equipment and the filter setting e Samples contain interfering substances e Troubleshoot if it interferes with the kit e Use of incompatible sample type e Refer data sheet to check if sample is compatible with the kit or optimization is needed e Sample readings above below the linear range e Concentrate Dilute sample so as
6. rol mix to background control wells Incubate for 30 min at room temperature protect from light Measure OD at 570 nm or fluorescence at E E 535 587nm Calculation Correct background by subtracting the value of the O Citrate Standard from all readings Next subtract the value of the Sample Background Control from the test sample Plot the standard curve Apply corrected sample readings to the standard curve to get Citrate amount in the sample wells The Citrate concentration in the test samples equals C Ay Sv nmol ul or umol ml or mM Where Ay is the amount of citrate nmol in your sample from the standard curve Sv is the sample volume ul added to the sample well Citric acid molecular weight 191 g mol a 10000 7500 535 587nm 5000 2500 y 0 126x 0 0131 RFU Ex Em y 8828 7x 698 65 0 0 25 0 5 0 75 1 Citrate nmol well Citrate nmol well Citrate standard curve generated using this kit protocol RELATED PRODUCTS ADP ATP Kit NAD NADH and NADP NADPH Assay Kits CoA Acetyl CoA Assay Kits Malic Acid Assay Kit a Ketoglutarate Assay Kit Malic Acid Assay Kit Isocitrate Assay Kit Starch Assay Kit Pyruvate Assay Kit Tel 408 493 1800 e Fax 408 493 1801 Email tech biovision com Pyruvate Assay Kit Glutamate Assay Kit Lactate Assay Kits Oxaloacetate Assay Kit Glycogen Assay Kit Glucose Assay Kit Maltose Assay Kit e www biovision com Page 1 of 2 BioVision re GEN
7. 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 Research Products Tel 408 493 1800 e Fax 408 493 1801 e www biovision com 155 South Milpitas Boulevard Milpitas CA 95035 Email tech biovision com Page 2 of 2
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