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 .
