Isocitrate Dehydrogenase Activity Assay Kit Catalog Number MAK062 Storage Temperature –20 C TECHNICAL BULLETIN Product Description Developer 1 vl Isocitrate dehydrogenase (IDH) catalyzes the Catalog Number MAK062E conversion of isocitrate to -ketoglutarate. In eukaryotes, there are three isozymes of IDH, the IDH Positive Control (NADP+) 20 L mitochondrial IDH2 and IDH3, and the cytoplasmic/ Catalog Number MAK062F peroxisomal IDH1. All three IDH family members require the presence of a divalent cation (Mg2+ or Mn2+) NADH Standard, 0.5 mole 1 vl and either the electron-accepting cofactor NADP+ (IDH1 Catalog Number MAK062G and IDH2) or NAD+ (IDH3) for their enzymatic activity. IDH1 and IDH2 mutations resulting in neomorphic Reagents and Equipment Required but Not enzymatic activity are found in certain cancers such as Provided. glioblastoma, acute myeloid leukemia, and colon 96 well flat-bottom plate – It is recommended to use cancer. This neoactivity shows a change in the clear plates for colorimetric assays. substrate specificity resulting in the conversion of Spectrophotometric multiwell plate reader -ketoglutarate to 2-hydroxyglutarate. Mutations in IDH family members are also associated with Ollier disease Precautions and Disclaimer and Maffucci syndrome. This product is for R&D use only, not for drug, household, or other uses. Please consult the Material The Isocitrate Dehydrogenase Activity Assay kit Safety Data Sheet for information regarding hazards provides a simple and direct procedure for measuring and safe handling practices. + + + NADP -dependent, NAD -dependent, or both NADP + and NAD -dependent IDH activity in a variety of Preparation Instructions samples. IDH activity is determined using isocitrate as Briefly centrifuge vials before opening. Use ultrapure the substrate in an enzyme reaction, which results in a water for the preparation of reagents. To maintain colorimetric (450 nm) product proportional to the reagent integrity, avoid repeated freeze/thaw cycles. enzymatic activity present. One unit of IDH is the amount of enzyme that will generate 1.0 mole of IDH Assay Buffer – Allow buffer to come to room NADH or NADP per minute at pH 8.0 at 37 °C. temperature before use. Components NAD+, NADP+, and IDH Substrate Buffer – Reconstitute The kit is sufficient for 100 assays in 96 well plates. each with 220 L of water. Mix well by pipetting, then aliquot and store, protected from light at IDH Assay Buffer 25 mL –20 C. Use within 2 months of reconstitution. Catalog Number MAK062A Developer – Reconstitute with 0.9 mL of water. Mix well + NAD 1 vl by pipetting (don’t vortex), then aliquot and store, Catalog Number MAK062B protected from light at –20 C. Use within + 2 months of reconstitution. NADP 1 vl Catalog Number MAK062C NADH Standard – Reconstitute in 50 L of water to generate a 10 mM NADH stock solution. Mix well IDH Substrate 1 vl by pipetting, then aliquot and store at –20 C. Use Catalog Number MAK062D within 2 months of reconstitution. 2 Storage/Stability Table 1. The kit is shipped on wet ice and storage at –20 C, Master Reaction Mixes protected from light, is recommended. Standards and Sample Reagent Procedure Samples Blank All samples and standards should be run in duplicate. IDH Assay Buffer 38 L 42 L Developer 8 L 8 L NADH Standards for Colorimetric Detection IDH Substrate 2 L – Dilute 10 L of the 10 mM NADH Standard with 90 L NAD+ and/or NADP+ 2 L – of the IDH Assay Buffer to prepare a 1 mM standard solution. Add 0, 2, 4, 6, 8, and 10 L of the 1 mM Note: For samples, if 2 L NAD+ is added, the assay standard solution into a 96 well plate, generating + + will detect NAD -dependent IDH. If 2 L NADP is 0 (blank), 2, 4, 6, 8, and 10 nmole/well standards. Add + added, the assay will detect NADP -dependent IDH. IDH Assay Buffer to each well to bring the volume to The addition of both 2 L NAD+ and 2 L NADP+ will 50 L. The NADH standard curve can be used as the + + detect total IDH activity. If both NAD and NADP are standard for the NAD+-dependent IDH as well as the + added, adjust the volume of IDH Assay Buffer to 36 L. NADP -dependent IDH activity. Sample Preparation 2. Add 50 L of the appropriate Master Reaction Mix 6 to each of the wells. Mix well using a horizontal Tissue (50 mg) or cells (1 10 ) can be homogenized shaker or by pipetting. in 200 L of ice-cold IDH Assay Buffer. Centrifuge the samples at 13,000 g for 10 minutes to remove 3. Incubate the plate at 37 C. After 2–3 minutes insoluble material. (T ), measure the absorbance at 450 nm initial (A ) . Serum samples can be directly added to wells. 450 initial Note: It is essential that (A ) is in the linear 450 initial range of the standard curve. Bring samples to a final volume of 50 L with IDH Assay Buffer. 4. Continue to incubate the plate at 37 C measuring Note: For unknown samples, it is suggested to test the absorbance (A ) every 5 minutes. Protect the several sample dilutions to ensure the readings are 450 plate from light during the incubation. within the linear range of the standard curve. 5. Continue taking measurements until the value of For the positive control (optional), add 2–5 L of the the most active sample is greater than the value of IDH positive control solution to wells and adjust to the highest standard (10 nmole/well). At this time 50 L with the IDH Assay Buffer. the most active sample is near or exceeds the end of the linear range of the standard curve. Assay Reaction 1. Set up the Master Reaction Mixes according to the 6. The final absorbance measurement [(A450)final] for scheme in Table 1. 50 L of the Master Reaction calculating the enzyme activity would be the Mix is required for each reaction (well). penultimate reading or the value before the most Note: NADPH and NADH in the samples will active sample is near or exceeds the end of the generate a background signal. To remove the effect linear range of the standard curve (see step 5). The of NADPH and NADH background, a sample blank time of the penultimate reading is Tfinal. may be set up for each sample by omitting the IDH + + Note: It is essential the final measurement falls substrate and NAD and/or NADP . within the linear range of the standard curve. 3 Results The IDH activity of a sample may be determined by the Calculations following equation: Correct for the background by subtracting the final measurement [(A450)final] obtained for the 0 (blank) IDH Activity = B Sample Dilution Factor NADH standard from the final measurement [(A450)final] (Reaction Time) V of the standards and samples. Background values can be significant and must be subtracted from all readings. B = Amount (nmole) of NADH generated between Tinitial Plot the NADH standard curve. and Tfinal. Note: A new standard curve must be set up each time Reaction Time = Tfinal – Tinitial (minutes) the assay is run. V = sample volume (mL) added to well Calculate the change in absorbance from Tinitial to Tfinal IDH activity is reported as nmole/min/mL = milliunit/mL. for the samples. One unit of IDH is the amount of enzyme that will generate 1.0 mole of NADH or NADP per minute at A450 = (A450)final – (A450)initial pH 8.0 at 37 C. Compare the A450 of each sample to the standard Example: curve to determine the amount of NADH and/or NADPH NADH (B) = 5.84 nmole (B) generated by the isocitrate dehydrogenase between First reading (Tinitial) = 3 minute Tinitial and Tfinal. Second reading (Tfinal) = 32 minutes Sample volume (V) = 0.01 mL Sample dilution is 1 IDH activity is: 5.84 1 = 20.14 milliunits/mL (32–3) 0.01 4 Troubleshooting Guide Problem Possible Cause Suggested Solution Cold assay buffer Assay Buffer must be at room temperature Omission of step in procedure Refer and follow Technical Bulletin precisely Assay not working Plate reader at incorrect wavelength Check filter settings of instrument Type of 96 well plate used For colorimetric assays, use clear plates Samples prepared in different buffer Use the Assay Buffer provided Repeat the sample homogenization, Cell/Tissue culture samples were increasing the length and extent of incompletely homogenized homogenization step. Samples with erratic Samples used after multiple freeze-thaw Aliquot and freeze samples if needed to use readings cycles multiple times Presence of interfering substance in the If possible, dilute sample further sample Use of old or inappropriately stored Use fresh samples and store correctly until samples used Thaw all components completely and mix Improperly thawed components gently before use Use of expired kit or improperly stored Check the expiration date and store the Lower/higher reagents components appropriately readings in samples Allowing the reagents to sit for extended Prepare fresh Master Reaction Mix before and standards times on ice each use Refer to Technical Bulletin and verify correct Incorrect incubation times or temperatures incubation times and temperatures Incorrect volumes used Use calibrated pipettes and aliquot correctly Thaw and resuspend all components before Use of partially thawed components preparing the reaction mix Pipetting errors in preparation of standards Avoid pipetting small volumes Prepare a Master Reaction Mix whenever Pipetting errors in the Reaction Mix possible Non-linear standard Pipette gently against the wall of the plate Air bubbles formed in well curve well Standard stock is at incorrect Refer to the standard dilution instructions in concentration the Technical Bulletin Recheck calculations after referring to Calculation errors Technical Bulletin Substituting reagents from older kits/lots Use fresh components from the same kit Samples measured at incorrect Check the equipment and filter settings wavelength Unanticipated results Samples contain interfering substances If possible, dilute sample further Sample readings above/below the linear Concentrate or dilute samples so readings range are in the linear range SS,LS,MAM 08/18-1 2018 Sigma-Aldrich Co.