ab83389 Glutamate Assay Kit Instructions for Use For the rapid, sensitive and accurate measurement of Glutamate in various samples. This product is for research use only and is not intended for diagnostic use.
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Table of Contents 1. Overview 3 2. Protocol Summary 4 3. Components and Storage 5 4. Assay Protocol 7 5. Data Analysis 9 6. Troubleshooting 10 2
1. Overview Glutamate, one of the two acidic proteinogenic amino acids, is also a key molecule in cellular metabolism. In humans, glutamate plays an important role both in amino acid degradation and disposal of excess or waste nitrogen. Glutamate is the most abundant swift excitatory neurotransmitter in the mammalian nervous system. It is believed to be involved in learning and memory and has appeared to be involved in diseases like amyotrophic lateral sclerosis, lathyrism, autism, some forms of mental retardation and Alzheimer's disease. Glutamic acid is also present in a wide variety of foods, and has been used as a flavor enhancer in food industry. Abcam s Glutamate Assay Kit provides a sensitive detection method of the glutamate in a variety of samples. The glutamate Enzyme Mix recognizes glutamate as a specific substrate leading to proportional color development. The amount of glutamate can therefore be easily quantified by colorimetric (spectrophotometry at λ = 450 nm) method. 3
2. Protocol Summary Sample Preparation Standard Curve Preparation Add Reaction Mix Measure Optical Density 4
3. Components and Storage A. Kit Components Item Quantity Glutamate Assay Buffer 25 ml Glutamate Enzyme Mix 1 vial Glutamate Developer 1 vial Glutamate Standard (0.1M) 0.1 ml * Store the kit at -20 C, protect from light. Allow Assay Buffer to warm to room temperature before use. Keep the Glutamate Enzyme Mix on ice during the assay and protect from light. Briefly centrifuge vials before opening. Read the entire protocol before performing the assay. 5
B. Additional Materials Required Microcentrifuge Pipettes and pipette tips Colorimetric microplate reader 96 well plate Orbital shaker 6
4. Assay Protocol 1. Sample Preparation: a. For tissues or cell samples: Tissues or cells (1 10 6 ) can be homogenized in 100 μl Assay Buffer. Centrifuge to remove insoluble material at 13,000 x g for 10 minutes. b. For serum samples: 10-50 μl serum samples can be directly diluted in the Assay Buffer. Bring sample wells to 50 μl/well with Assay Buffer in a 96-well plate. Prepare a parallel sample well as the background control. We suggest testing several doses of your sample to make sure the readings are within the standard curve range. 2. Standard Curve Preparation: Dilute 10 μl of the 0.1M Glutamate standard with 990 μl Assay Buffer to generate 1 mm standard Glutamate. Add 0, 2, 4, 6, 8, 10 μl of the diluted Glutamate standard into a 96-well plate in duplicate to generate 0, 2, 4, 6, 8, 10 nmol/well standard. Bring the volume to 50 μl with Assay buffer. 3. Glutamate Reaction Mix: a) Reconstitute Glutamate Enzyme Mix with 220 μl Assay Buffer. Aliquot enough Glutamate Enzyme Mix (2 μl per assay) for the number of assays to be performed in each experiment and freeze the stock solution immediately at -20 C for future use. 7
The Glutamate Enzyme Mix is stable for up to 2 months at -20 C after reconstitution or freeze-thaw cycle less than 5 times. b) Reconstitute Glutamate Developer with 820 μl of ddh 2 O. Pipette up and down several times to completely dissolve the pellet into solution (Don t vortex). c) Mix enough reagents for the number of samples and standards to be performed. For each well, prepare a total 100 μl Reaction Mix: Reaction Mix Bkgd Control Mix Assay Buffer 90 µl 92 µl Glutamate Developer 8 µl 8 µl Glutamate Enzyme Mix 2 µl -- Add 100 μl of the Reaction Mix to each well containing the Glutamate Standard and test samples. To the background control well, add 100 µl of background control mix. Mix well. Incubate at 37 C for 30 minutes, protect from light. 4. Measure OD at 450 nm in a microplate reader. 8
5. Data Analysis Correct background by subtracting the value derived from the background control from all sample readings (The background reading can be significant and must be subtracted from sample readings). Plot Glutamate standard curve. Glutamate concentrations of the test samples can then be calculated: Concentration = Sa / Sv nmol/μl, or mm Where: Sa is the sample amount of unknown (in nmol) from standard curve, Sv is sample volume (μl) added into the wells. L-Glutamic acid Molecular Weight is 147.13 g/mol. 9
6. Troubleshooting Problem Reason Solution Assay not working Unexpected results Assay buffer at wrong temperature Protocol step missed Plate read at incorrect wavelength Unsuitable microtiter plate for assay Measured at wrong wavelength Samples contain impeding substances Unsuitable sample type Sample readings are outside linear range Assay buffer must not be chilled - needs to be at RT Re-read and follow the protocol exactly Ensure you are using appropriate reader and filter settings (refer to datasheet) Fluorescence: Black plates (clear bottoms); Luminescence: White plates; Colorimetry: Clear plates. If critical, datasheet will indicate whether to use flat- or U-shaped wells Use appropriate reader and filter settings described in datasheet Troubleshoot and also consider deproteinizing samples Use recommended samples types as listed on the datasheet Concentrate/ dilute samples to be in linear range 10
Samples with inconsistent readings Lower/ Higher readings in samples and standards Unsuitable sample type Samples prepared in the wrong buffer Samples not deproteinized (if indicated on datasheet) Cell/ tissue samples not sufficiently homogenized Too many freezethaw cycles Samples contain impeding substances Samples are too old or incorrectly stored Not fully thawed kit components Out-of-date kit or incorrectly stored reagents Reagents sitting for extended periods on ice Incorrect incubation time/ temperature Incorrect amounts used Problem Reason Solution Refer to datasheet for details about incompatible samples Use the assay buffer provided (or refer to datasheet for instructions) Use the 10kDa spin column (ab93349) Increase sonication time/ number of strokes with the Dounce homogenizer Aliquot samples to reduce the number of freeze-thaw cycles Troubleshoot and also consider deproteinizing samples Use freshly made samples and store at recommended temperature until use Wait for components to thaw completely and gently mix prior use Always check expiry date and store kit components as recommended on the datasheet Try to prepare a fresh reaction mix prior to each use Refer to datasheet for recommended incubation time and/ or temperature Check pipette is calibrated correctly (always use smallest volume pipette that can pipette entire volume) 11
Standard curve is not linear Not fully thawed kit components Pipetting errors when setting up the standard curve Incorrect pipetting when preparing the reaction mix Air bubbles in wells Concentration of standard stock incorrect Errors in standard curve calculations Use of other reagents than those provided with the kit Wait for components to thaw completely and gently mix prior use Try not to pipette too small volumes Always prepare a master mix Air bubbles will interfere with readings; try to avoid producing air bubbles and always remove bubbles prior to reading plates Recheck datasheet for recommended concentrations of standard stocks Refer to datasheet and re-check the calculations Use fresh components from the same kit For further technical questions please do not hesitate to contact us by email (technical@abcam.com) or phone (select contact us on www.abcam.com for the phone number for your region). 12
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