ab83431 Alpha Ketoglutarate (alpha KG) Assay Kit Instructions for Use For the rapid, sensitive and accurate measurement of alpha Ketoglutarate 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 11 2
1. Overview Alpha Ketoglutarate (alpha KG) is a key intermediate in the Krebs cycle, coming after isocitrate and before succinyl CoA. Anaplerotic reactions replenish the cycle by synthesizing alpha KG from transamination of glutamate, or through the action of glutamate dehydrogenase. Alpha KG is an important nitrogen transporter. Being a key intermediate, it is one of the organic acids measured in newborns as an indicator of inborn errors of metabolism. Abcam's Alpha Ketoglutarate (alpha KG) Assay Kit provides a simple, sensitive and rapid means for quantifying alpha KG in a variety of samples. In the assay, alpha Ketoglutarate is transaminated with the generation of pyruvate which is utilized to convert a nearly colorless probe to both color (λ max = 570 nm) and fluorescence (Ex/Em = 535/587 nm). The Alpha Ketoglutarate Assay Kit is useful for detecting alpha Ketoglutarate in the range of 0.01-10 nmoles. 3
2. Protocol Summary Sample Preparation Standard Curve Preparation Prepare and Add Reaction Mix Measure Optical Density or Fluorescence 4
3. Components and Storage A. Kit Components Item Quantity alpha KG Assay Buffer 25 ml alpha KG OxiRed Probe (in DMSO) 0.2 ml alpha KG Converting Enzyme (Lyophilized) 1 vial alpha KG Development Enzyme Mix (Lyophilized) 1 vial alpha KG Standard (10 µmol; Lyophilized) 1 vial * Store kit at -20 C, protect from light. Warm alpha KG Assay Buffer to room temperature before use. Briefly centrifuge all small vials prior to opening. ALPHA KG PROBE: Ready to use as supplied. Warm to room temperature before using to melt frozen DMSO. Protect from light and moisture. Stable for 2 months at -20 C. ALPHA KG CONVERTING ENZYME, ALPHA KG ENZYME MIX: Dissolve with 220 µl alpha KG Buffer separately. Pipette up and 5
down to dissolve. Aliquot into vials with a sufficient amount for each experiment and store at -20 C. Avoid repeated freeze/thaw cycles. Use within two months. ALPHA KG STANDARD: Dissolve in 100 µl dh 2 O to generate 100 mm (100nmol/µl) alpha KG Standard solution. Keep cold while in use. Store at -20 C. B. Additional Materials Required Microcentrifuge Pipettes and pipette tips Fluorescent or colorimetric microplate reader 96 well plate Orbital shaker 6
4. Assay Protocol 1. Sample Preparation: Tissue (20 mg) or cells (2 x 10 6 ) are rapidly homogenized with 100 µl of ice cold PBS or other buffer (ph 6.5-8). Note: Enzymes in samples may interfere with the assay. We suggest deproteinizing samples using 10 kda molecular weight cut off spin columns (ab93349) or using the perchloric acid/koh protocol as follows: a) Tissue samples (20-1000 mg) should be frozen rapidly (liquid N 2 or methanol/dry ice), weighed and pulverized. b) Add 2 µl 1N perchloric acid/mg per sample. KEEP COLD! c) Homogenize or sonicate thoroughly. Spin homogenate at 10,000 x g for 5-10 minutes. d) Neutralize supernatant with 3M KHCO 3, adding repeated 1 µl aliquots/10 µl supernate while vortexing. Add until bubble evolution ceases (2-5 aliquots). Put on ice for 5 minutes. e) Check ph (using 1 µl) is ~6-8. Spin 2 minutes at 10,000 x g to pellet KClO 4. Add 1-50 µl samples into duplicate wells of a 96-well plate and bring volume to 50 µl with Assay Buffer. 7
We suggest testing several doses of your samples to ensure readings are within the linear range. 2. Standard Curve Preparation: a. For the colorimetric assay: Dilute the alpha KG Standard to 1 nmol/µl by adding 10µl of the Standard to 990 µl of dh 2 O, mix well. Add 0, 2, 4, 6, 8, 10 µl into a series of standards wells on a 96-well plate. Adjust volume to 50 µl/well with Assay Buffer to generate 0, 2, 4, 6, 8, 10 nmol/well of the Standard. b. For the fluorometric assay: Dilute further by adding 10 µl to 90 µl of dh 2 O. Add 0, 2, 4, 6, 8, 10 µl into a series of standards well on a 96-well plate. Adjust the volume to 50 µl/well to generate 0, 0.2, 0.4, 0.6, 0.8, 1.0 nmol/well. 3. Alpha KG Reaction Mix: Mix enough reagent for the number of samples and standards to be performed: For each well, prepare a total 50 µl Reaction Mix containing: Sample Bkgd Control* Alpha KG Assay Buffer 44 µl 46 µl Alpha KG Converting Enzyme 2 µl --- Alpha KG Enzyme Mix 2 µl 2 µl Alpha KG Probe 2 µl 2 µl 8
* Note: Pyruvate generates background. If pyruvate is suspected in your sample, you can do the background control omitting the converting enzyme. The background control can be subtracted from the alpha KG reading. Note (optional): For fluorometric assay use 0.4 µl Pyruvate Probe and 45.6 µl (47.6 µl for Bkgd Control) Pyruvate assay buffer to reduce background 4. Add 50 µl of the Reaction Mix to each well containing the alpha KG Standard, samples or background control.* 5. Incubate for 30 min at 37 C, protect from light. 6. Measure OD at 570 nm or fluorescence using Ex/Em = 535/587 nm. 5. Data Analysis Correct background by subtracting the value of the zero alpha KG blank from all readings. Plot the standard curve. Apply the corrected sample readings to the standard curve to get alpha KG amount in the sample wells. The alpha KG concentrations in the test samples: Concentration = Ay / Sv (nmol/µl; or µmol/ml; or mm) 9
Where: Ay is the amount of alpha KG (nmol) in your sample from the standard curve. Sv is the sample volume (µl) added to the sample well. Alpha Ketoglutarate molecular weight: 146.11 Alpha Ketoglutarate standard curve generated using this kit protocol. 10
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 11
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 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) 12
Problem Reason Solution 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). 13
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