SAMfluoro™: SAM Methyltransferase Assay
Standard Assay: Label 8 clean glass test tubes with A-H and prepare the standards as indicated below.
The diluted standards are stable for 4 hours at room temperature.
Tube10µM Resorufin Stock (µl)1X Resorufin Buffer (µl)Final Concentration (µM)A1258751.25B2507502.5C3756253.75D5005005E6253756.25F7502507.5G8751258.75H1000010.    
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.
Positive Control: Adenosylhomocysteine: The vial contains 200µl of a 1mM solution of adenosylhomocysteine (AdoHcy).
Thaw the vial on ice.
Prior to use, dilute 10µl with 90µl of SAM Methyltransferase Assay Buffer with Additive.
SAMfluoro Enzyme Mix, supplied in 300µl vials.
Each vial is suitable for 36 assays.
Thaw on ice only the number of vials you require for your assay.
S-Adenosylmethionine (SAM), supplied lyophilized.
Reconstitute the contents ofthe vial with 100µl HCl Assay Reagent [20mM] to yield 6.9mM SAM.
SAMfluoro Fluorometric Mix.
Immediately prior to making the Master Mix, add 100µl DMSO to the vial and vortex.
Add 400µl proteomic grade or ultra pure water and vortex.
Resorufin Buffer: Add 4ml Resorufin Buffer [10X] to 36ml ultra pure water to generate 1X Resorufin Buffer.
Sample Preparation: Prepare your test sample, containing the purified SAM dependent methyltransferase to be assayed, according to your own standard protocol.
Prepare the specific substrate for the methyltransferase to be assayed using the SAM Methyltransferase Assay Buffer or the buffer of your own choice.
Standard Assay: Add 115µl of each standard per well of a fluorescent compatible plate.
Perform in duplicate.
Standard Assay: Blank the plate against 1X Resorufin Buffer.
Read the plate after 5 minutes using an excitation wavelength of 530-540nm and an emission wavelength of 585-595nm.
Equilibrate the SAM Methyltransferase Assay Buffer + Additive to 37 °C.
Aliquot a total volume of 5μl 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, pH8.0 as a diluent.
We recommend performing the reactions and controls in at least duplicate.
a. For the background control, aliquot 5μl SAM Methyltransferase Assay Buffer into each background control well.
We recommend performing the reactions in duplicate.
b. For the positive control, add 5μl Positive Control and 10μl SAM Methyltransferase Assay Buffer to each positive control well.
We recommend performing the reactions in duplicate.
Add 10µl the appropriate acceptor substrate to the sample and background control wells, using SAM Methyltransferase Assay Buffer or 0.1M Tris, pH8.0 as a diluent.
Immediately prior to use and in a suitable tube, prepare the SAM Methyltransferase Assay Master Mix according to the table below:
Reagent36 wells72 wells100 wellsSAM Methyltransferase Assay Buffer + Additive3ml6ml9mlSAMfluoro Enzyme Mix1 vial/ 300µl2 vial/ 600µl3 vial/ 900µlSAMfluoro Fluorometric Mix200µl400µl600µlS-Adenosylmethionine1 vial/ 100µl2 vials/ 200µl3 vials/ 300µl.  
Immediately initiate the reaction by adding 100μl SAM Methyltransferase Master Mix to the wells.
Immediately, read the plate at 37°C every minute for 30 minutes using an excitation wavelength of 530-540nm and an emission wavelength of 585- 595nm.
Standard Assay: Dilute 25µl resorufin standard with 475µl 1X Resorufin Buffer.
Standard Assay: Mix 500µl of this diluted standard with 4.5ml 1X Resorufin to yield a stock solution of 10µM.
