CBA096 SAM Methyltransferase Assay

CBA096
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      Overview

      Replacement Information

      Key Specifications Table

      Detection Methods
      Colorimetric

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      CBA096-1KIT
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          Description
          OverviewMethylation of biological molecules and proteins plays an important role in signal transduction, biosynthesis, protein repair, gene silencing and chromatin regulation. The SAM Methyltransferase Assay is a continuous enzyme coupled assay that can monitor SAM dependent methyltransferases without the use of radioactive labels or endpoint measurements.
          Catalogue NumberCBA096
          Brand Family Calbiochem®
          Application Data



          Left Panel: Assay using the Adenosylhomocysteine positive control. Right Panel: Human lysine specific histone methyltransferase SET7/9 was measured as outlined in the Detailed Protocol using 20 µM TAF-10 as the acceptor substrate.
          Materials Required but Not Delivered SAM Methyltransferase to be tested
          Appropriate methyltransferase acceptor substrate
          References
          ReferencesDorgan, K.M., et al. 2006. Anal. Biochem. 350, 249.
          Shubert, H.L., et al. 2003. Trends Biochem. Sci. 28, 329.
          Cheng, X. and Blumentahl, R.M. 1999. S-Adenosylmethionine Dependent Methyltransferases: Structures and Functions World Scientific, Singapore.
          Product Information
          Detection methodColorimetric
          Form100 Tests
          Format96-well plate
          Kit containsSAM Methyltransferase Assay Buffer, Adenosylhomocysteine, Methyltransferase Enzyme Mix, SAM Methyltransferase Assay Buffer additive, SAM Colorimetric Mix
          Applications
          Biological Information
          Assay range0.013-0.133 µmol/min/ml
          Physicochemical Information
          Dimensions
          Materials Information
          Toxicological Information
          Safety Information according to GHS
          Safety Information
          R PhraseR: 36/37/38

          Irritating to eyes, respiratory system and skin.
          S PhraseS: 26-36-45

          In case of contact with eyes, rinse immediately with plenty of water and seek medical advice.
          Wear suitable protective clothing.
          In case of accident or if you feel unwell, seek medical advice immediately (show the label where possible).
          Product Usage Statements
          Intended useThe SAM Methyltransferase Assay is a continuous enzyme coupled assay that can continuously monitor SAM dependent methyltransferases without the use of radioactive labels or endpoint measurements.
          Storage and Shipping Information
          Ship Code Dry Ice Only
          Toxicity Irritant
          Storage ≤ -70°C
          Storage ConditionsUpon arival store the entire contents of the kit at -70°C.
          Do not freeze Ok to freeze
          Packaging Information
          Transport Information
          Supplemental Information
          Kit containsSAM Methyltransferase Assay Buffer, Adenosylhomocysteine, Methyltransferase Enzyme Mix, SAM Methyltransferase Assay Buffer additive, SAM Colorimetric Mix
          Specifications

          Documentation

          SAM Methyltransferase Assay Certificates of Analysis

          TitleLot Number
          CBA096

          References

          Reference overview
          Dorgan, K.M., et al. 2006. Anal. Biochem. 350, 249.
          Shubert, H.L., et al. 2003. Trends Biochem. Sci. 28, 329.
          Cheng, X. and Blumentahl, R.M. 1999. S-Adenosylmethionine Dependent Methyltransferases: Structures and Functions World Scientific, Singapore.
          User Protocol

          Revision21-June-2013 JSW
          Form100 Tests
          Format96-well plate
          Detection methodColorimetric
          StorageUpon arival store the entire contents of the kit at -70°C.
          Intended useThe SAM Methyltransferase Assay is a continuous enzyme coupled assay that can continuously monitor SAM dependent methyltransferases without the use of radioactive labels or endpoint measurements.
          BackgroundMethylation of key biological molecules and proteins plays an important role in numerous biological systems, including signal transduction, biosynthesis, protein repair, gene silencing, and chromatin regulation. The S-adenosylmethionine (SAM) dependent methyltransferases use SAM, the second most commonly used enzymatic cofactor after ATP. SAM, also known as AdoMet, acts as a donor of a methyl group that is required for the modification of proteins and DNA. Aberrant levels of SAM have been linked to many abnormalities, including Alzheimer's, depression, Parkinson's, multiple sclerosis, liver failure and cancer.
          Principles of the assay

          Figure 1: Transmethylation


          The removal of the methyl group from SAM generates S-adenosylhomocysteine, which is rapidly converted to S-ribosylhomocysteine and adenine by the included adenosylhomocysteine nucleosidase. This rapid conversion prevents the buildup of adenosylhomocysteine and its feedback inhibition on the methylation reaction. Finally, the adenine is converted to hypoxanthine, by adenine deaminase, which in turn is converted to urate and hydrogen peroxide. The rate of production of hydrogen peroxide is measured with a colorimetric assay by an increase in absorbance at 510 nm. The assay is supplied with adenosylhomocysteine as a positive control. The assay can be adapted to be used with any SAM dependent methyltransferase or an enzyme reaction that produces 5-adenosylhomocysteine or 5'-methylthioadenosine, due to the specificity of adenosylhomocysteine nucleosidase.
          Materials provided• SAM Methyltransferase Assay Buffer (Kit Component No. JA9529-20ML): 1 bottle, 20 ml
          • Adenosylhomocysteine (Kit Component No. JA9530-0.2ML): 1 vial, 200 µl
          • S-Adenosylmethionine (Kit Component No. JA9534-1EA): 3 vials, lyophilized
          • SAM: Methyltransferase Enzyme Mix (Kit Component No. JA9531-300UL): 3 vials, 300 µl each
          • Methyltransferase Assay Buffer Additive (Kit Component No. JA9532-0.2ML): 1 vial, 0.2 ml
          • Colorimetric Mix (Kit Component No. JA9533-1EA): 1 vial, lyophilized
          • HCl Assay Reagent (Kit Component No. 5.05411.0002): 1 vial, 1 ml, 20 mM
          Materials Required but not provided SAM Methyltransferase to be tested
          Appropriate methyltransferase acceptor substrate
          Precautions and recommendations The positive control, Adenosylhomocysteine, supplied is a control for the SAM Methyltransferase Assay. Some acceptor substrates, inhibitors, activators or buffer components may interfere with the assay. We highly recommend testing the compatibility of these reagents with the SAM Methyltransferase Assay using the positive control, by substituting in suspected non compatible reagent into the positive control reactions. It is necessary to titrate each enzyme/substrate system in the assay to determine optimal conditions.
          The deaminase in the SAM methyltransferase assay requires the presence of divalent metal ions, present in the SAM Methyltransferase Assay Buffer. The presence of chelators, such as EDTA, will deplete divalent metal ions and inhibit the deaminase enzyme. If EDTA is required then supplement additional manganese ions into the reaction.
          Reducing agents, including DTT, β-mercaptoethanol and TCEP, have an inhibitory effect on the assay. If present, we recommend dialysis against 0.1M Tris, pH8.0.
          Detergents: at some concentrations they appear to improve absorbance, but this is most likely due to the presence of peroxides in the detergents, which will interfere with the assay.
          Preparation Prepare your test sample, containing the SAM dependent methyltransferase to be assayed, according to your own standard protocol. Avoid the use of reducing agents and metal chelators as these have an inhibitory effect on the reaction.
          Reagent preparation• SAM Methyltransferase Assay Buffer with Additive: Thaw SAM Methyltransferase Assay Buffer and SAM Methyltransferase Assay Buffer Additive solution at room temperature. Add the entire volume of SAM Methyltransferase Assay Buffer Additive into the SAM Methyltransferase Assay Buffer and mix it thoroughly. Store SAM Methyltransferase Assay Buffer at room temperature, do not freeze after addition of Additive. • Methyltransferase Enzyme Mix: Thaw on ice. Following initial thaw, dispense into 150 µl aliquots (enough for 10 assays) and store at -70°C. Each assay requires 15 µl. SAM Colorimetric Mix: Add 1 ML SAM Methyltransferase Assay Buffer with Additive, wait 5 min, and then vortex to dissolve. Store reconstituted SAM Colorimetric Mix at -70°C. • Substrate: Prepare specific substrate for the methyltransferase to be assayed using the SAM Methyltransferase Assay Buffer or the buffer of your own choice.
          S-Adenosylmethionine: Reconsitute the contents of the vial with 100 µl HCl Assay Reagent to yeild 6.9 mM S-Adenosylmethionine. Each vial is suitable for 36 assays. We do not recommend repeated freeze/thawing of the S-Adenosylmethionine solution.
          Detailed protocolNote: The positive control supplied is a control for the SAM Methyltransferase Assay. Some acceptor substrates, inhibitors, activators or buffer components may interfere with the assay. We highly recommend testing the compatibility of these reagents with the SAM Methyltransferase Assay using the positive control, by substituting in suspected non compatible reagent into the positive control reactions. It is necessary to titrate each enzyme/ substrate system in the assay to determine optimal conditions.

          NOTE: The deaminase in the SAM methyltransferase assay requires the presence of divalent metal ions, present in the SAM Methyltransferase Assay Buffer. The presence of chelators, such as EDTA, will deplete divalent metal ions and inhibit the deaminase enzyme. If EDTA is required then supplement additional manganese ions into the reaction.

          NOTE: Reducing agents, including DTT, β-mercaptoethanol and TCEP, have an inhibitory effect on the assay. If present, we recommend dialysis against 0.1M Tris-HCl, pH 8.0.

          1. Equilibrate the SAM Methyltransferase Assay Buffer + Additive to 37°C.
          NOTE: The SAM Buffer + Additive must be at 37°C prior to performing the assay. Failure to prewarm will result in artifactual results.
          2.Aliquot a total volume of 5 µl of your SAM methyltransferase samples to at least two wells of a 96 well plate. Use the SAM Methyltransferase Assay Buffer or 0.1M Tris, pH 8.0 as a diluent. We recommend performing the reactions and controls in at least duplicate.
          a. For the background control, aliquot 5 µl SAM Methyltransferase Assay Buffer into each background control well. We recommend performing the reactions in duplicate.
          b. For the positive control, add 5 µl Positive Control and 10 µl SAM Methyltransferase Assay Buffer to each positive control well. We recommend performing the reactions in duplicate.
          3. Add 10 µl of the appropriate acceptor substrate to the sample and background control wells, using SAM Methyltransferase Assay Buffer or 0.1M Tris, pH 8.0 as a diluent.
          NOTE: If assaying inhibitors or activators, adjust the acceptor substrate concentration so that the substrate and activators or inhibitors are added in a final volume of 10 µl.
          4. Immediately prior to use and in a suitable tube, prepare the SAM Methyltransferase Assay Master Mix according to the table below:

          Table 1: Assay Mix



          5. Immediately initiate the reaction by adding 100 µl SAM Methyltransferase Master Mix to the wells. Immediately, zero the wells and begin measuring the absorbances at 510nm collecting data every 10-30 seconds at 37°C until the increasing absorbances plateau (approx. 15-30 mins).
          6. Subtract the background control from your samples and positive control and then plot a curve of time (min) against absorbance. The rate of the enzyme activity can be characterized by the absorbance increase per min.
          Calculations1. Calculate the average absorbance of each sample.
          2. Calculate the change in absorbance (ΔAbs) per min by:
          a. Plot the absorbances against time to obtain the slope (rate) of the linear portion of the curve. See figure below for a plot with the Adenyosylhomocysteine (left pane) and human lysine specific histone methyltransferase, SET7/9 (right panel). Subtract the background control from each sample and positive control and plot a curve of time (min) against absorbance. The rate of the enzyme activity can be characterized by the absorbance increase per min.
          OR
          b. Calculate the change in absorbance between two points on the linear portion of the curve using the following equation:

          Figure 2: Calculate the Change in Absorbance Between Two Points


          3. Calculate the rate of ΔAbs/min for the background control wells and subtract this rate from each sample rate.
          4. Calculate the methyltransferase activity: Use the following equation to calculate the methyltransferase activity:

          Figure 3: Calculate the Methyltransferase Activity


          The rate of the reaction is determined using the 3,5-dichloro-2-hydroxybenzenesulfonic acid (DHBS) extinction coeffieicnet of 15.0 mM*. One unit of methyltransferase will transfer 1 µmol of a methyl group per min at 37°C. *Note: The actual extinction coefficient of 3,5-dichloro-2-hydroxybenzenesulfonic acid (DHBS) is 26.0 mM-1cm-1. This value has been adjusted for the pathlength of the solution in the well (0.577 cm). 5. If activators or inhibitors were assayed, determine the percent activation/inhibition for each sample as follows:

          Figure 4: Determine the Percent Activation or Inhibition


          Example data

          Figure 5: Methyltransferase Activity

          Left Panel: Assay using the Adenosylhomocysteine positive control. Right Panel: Human lysine specific histone methyltransferase SET7/9 was measured as outlined in the Detailed Protocol using 20 µM TAF-10 as the acceptor substrate.

          Assay Range0.013-0.133 µmol/min/ml
          Assay Range NotesThe assay range is 0.013-0.133 µmol/min/ml of methytransferase activity, which is equivalent to an absorbance range of 0.01-0.1 per min.
          Registered TrademarksCalbiochem® is a registered trademark of , KGaA, Darmstadt
          Interactive Pathways™ is a trademark of , KGaA, Darmstadt