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Description
Overview
Methylation 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 Number
CBA096
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
References
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.
Product Information
Detection method
Colorimetric
Form
60 Tests
Format
96-well plate
Kit contains
SAM Methyltransferase Assay Buffer, Adenosylhomocysteine, Methyltransferase Enzyme Mix, SAM Methyltransferase Assay Buffer additive, SAM Colorimetric Mix
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 use
The 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 Conditions
Upon arival store the entire contents of the kit at -70°C.
Do not freeze
Ok to freeze
Supplemental Information
Kit contains
SAM Methyltransferase Assay Buffer, Adenosylhomocysteine, Methyltransferase Enzyme Mix, SAM Methyltransferase Assay Buffer additive, SAM Colorimetric Mix
SAM Methyltransferase Assay Certificates of Analysis
Title
Lot 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
Revision
21-June-2013 JSW
Form
60 Tests
Format
96-well plate
Detection method
Colorimetric
Storage
Upon arival store the entire contents of the kit at -70°C.
Intended use
The 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.
Background
Methylation 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.
• 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 protocol
Note: 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.
Calculations
1. 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 Range
0.013-0.133 µmol/min/ml
Assay Range Notes
The 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 Trademarks
Calbiochem® is a registered trademark of Merck, KGaA, Darmstadt Interactive Pathways™ is a trademark of Merck, KGaA, Darmstadt
