Adenosylhomocysteinase (AHCY) Activity Fluorometric Assay Kit
Catalog Number EPI021 Storage Temperature –20 C
TECHNICAL BULLETIN
Product Description Adenosine Standard (10 mM) 100 L Adenosylhomocysteinase (AHCY, EC 3.3.1.1) or Catalog Number EPI021G S-adenosylhomocysteine hydrolase (SAHH); is an enzyme that catalyzes the reversible hydrolysis of S-adenosyl homocysteine (SAH) to adenosine and AHCY Enzyme (Positive Control) 10 L homocysteine. AHCY regulates the intracellular SAH Catalog Number EPI021H concentration which in turn regulates S-adenosyl methionine (SAM)-dependent methyltransferases. Reagents and Equipment Required but Not Down-regulation of AHCY has been associated with Provided. certain forms of cancer and Huntington’s disease, while 96 well flat-bottom plate – It is recommended to use in Wilson’s disease; the enzyme is inhibited by the accumulated copper. Mutations in the AHCY gene black plates for this assay. cause SAHH deficiency disease. Fluorescence multiwell plate reader
The AHCY Activity Assay kit can kinetically measure Precautions and Disclaimer AHCY activity by detecting adenosine generation This product is for R&D use only, not for drug, resulting from the hydrolysis of SAH. Adenosine is household, or other uses. Please consult the Safety detected via a multi-step reaction, resulting in the generation of an intermediate that reacts with the Data Sheet for information regarding hazards and safe Probe. The fluorescent product is measured at. Limit of handling practices. quantification (LOQ) is 1 U of recombinant human AHCY. Preparation Instructions Briefly centrifuge vials before opening. To maintain Components reagent integrity, avoid repeated freeze/thaw cycles. The kit is sufficient for 100 assays in 96 well plates. AHCY Assay Buffer – Bring to 37 C °C before use. AHCY Assay Buffer 25 mL Store at –20 C or 4 C. Catalog Number EPI021A Enzyme Mix I and Enzyme Mix II – Reconstitute with Homogenization Buffer 60 mL 210 L of Homogenization Buffer and mix gently by Catalog Number EPI021B pipetting. Briefly centrifuge to collect the contents in the bottom of the tube. Aliquot and store at –20 C. Avoid repeated freeze/thaw cycles. Probe 200 L
Catalog Number EPI021C AHCY Enzyme – Store at –20 C. Avoid repeated freeze/thaw cycles. SAH Substrate 100 L Catalog Number EPI021D Storage/Stability The kit is shipped on wet ice and storage at –20 C, Enzyme Mix I 1 vial protected from light, is recommended. Catalog Number EPI021E
Enzyme Mix II 1 vial Catalog Number EPI021F
2
Procedure 2. Prepare enough reagents for the number of assays All samples and standards should be run in duplicate. to be performed. Make 50 L of appropriate mix, see Table 1. Sample Preparation Rinse tissue and transfer 100 mg of fresh or frozen Table 1. tissue (stored at –70 C) to a prechilled tube. Add Preparation of Mixes 300 L of cold Homogenization Buffer containing protease inhibitor cocktail (not provided) and thoroughly Reagent Reaction Background homogenize tissue on ice. Transfer the tissue Mix Control Mix homogenate to a cold microfuge tube. AHCY Assay Buffer 44.5 L 45.5 L Enzyme Mix I 2 L 2 L To prepare cell extract, add 150–300 L of cold Enzyme Mix II 2 L 2 L Homogenization Buffer containing protease inhibitor Probe 6 0.5 L 0.5 L cocktail (not provided) to 1–5 10 fresh or frozen cells SAH Substrate 1 L – and pipette several times to disrupt the cells. Transfer cell homogenate including cell debris to a cold 3. Add 50 L of Reaction Mix into each Sample and microfuge tube and agitate on a rotary shaker at 4 C Positive Control wells and 50 L of Background for at least 15 minutes. Control mix to Standards and Sample Background
Control well(s). Mix well. Centrifuge the tissue or cell homogenate at 16,000 g 4. Measure fluorescence ( = 535 nm/ = 587 nm) for 10 minutes at 4 C. Transfer the clarified ex em in kinetic mode for at least 30 minutes. at 37 C. supernatant to a fresh pre-chilled tube and store on ice. Choose two time points (T and T ) in linear range Use lysates immediately to assay AHCY activity. 1 2 (can be as short as 2 minutes) of plot and obtain Note: Lysates can be aliquoted and snap frozen in corresponding RFU for sample (R and R ) and liquid nitrogen before storing at –20 C. Avoid S1 S2 sample Background Control (RBG1 and RBG2). Read freeze/thaw cycles. the Adenosine Standard Curve along with the samples. Adenosine Standard Dilute Adenosine Standard to 1 mM by adding 10 L of Results 10 mM Adenosine Standard to 90 L of AHCY Assay Calculations Buffer. Further dilute the Adenosine Standard to 10 M Subtract 0 Standard reading from all Standard and by adding 10 L of 1 mM Adenosine to 990 L of AHCY Positive Control Readings. Plot the Adenosine Assay Buffer. Add 0, 2, 4, 6, and 8 L of diluted 10 M Standard Curve. Subtract sample Background Control
Adenosine Standard into a series of wells in a 96 well reading from sample reading. Compare the RFU to plate to generate 0, 20, 40, 60, and 80 pmole/well the Standard Curve to obtain B (pmole of Adenosine) Adenosine Standard. Adjust the volume to 50 L/well generated by the sample during the reaction time with AHCY Assay Buffer. (T = T2 – T1).
AHCY Activity Assay AHCY Activity = B 1. Add 2–50 L (5–25 g protein) of cell/tissue (T) g of protein homogenate or purified protein into 96 well plate. Dilute AHCY enzyme (Positive Control) as needed, pmole/min/g = U/g = mU/mg (nmoles/min 1:10 in AHCY Assay Buffer just before use. Use adenosine generated per mg of protein) 1–4 L of diluted AHCY enzyme for the assay. Bring the volume of samples and Positive Control B = Adenosine amount from the Standard Curve to 50 L/well with AHCY Assay Buffer. (pmole) Notes: For unknown samples, testing several doses T = reaction time (minutes) to ensure the readings are within the Standard g of protein = amount of protein/well (g) Curve range is suggested. Unit Definition: One unit of AHCY activity is the amount For samples having adenosine background, of enzyme that hydrolyzes the substrate to yield 1.0 prepare parallel sample well(s) as sample mol of adenosine/minute at 37 C. background control(s). 3
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 Black plates are recommended for this assay Use the Assay Buffer provided or refer to Samples prepared in different buffer Technical Bulletin for instructions 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 samples will be readings cycles used 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 use 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 Reaction Mix before each use and standards times on ice 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 Pipetting errors in the Reaction Mix Prepare a Reaction Mix whenever possible Pipette gently against the wall of the plate Non-linear standard 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
SJ,MAM 05/16-1
2016 Sigma-Aldrich Co. LLC. All rights reserved. SIGMA-ALDRICH is a trademark of Sigma-Aldrich Co. LLC, registered in the US and other countries. Sigma brand products are sold through Sigma-Aldrich, Inc. Purchaser must determine the suitability of the product(s) for their particular use. Additional terms and conditions may apply. Please see product information on the Sigma-Aldrich website at www.sigmaaldrich.com and/or on the reverse side of the invoice or packing slip.