Arthus Biosystems -Model FK00201 -Flow Cytometry Assay Kit 1 for S-Adenosylmethionine (SAM)

Catalog No. FK00201

Packaging size 100 tests

1. Prepare the cells to be tested in the amount of 5 -10 x 105/sample in Eppendorf tubes.
2. Centrifuge the cell suspension at 1500 rpm for 5 min, and discard the supernatant.
3. Fix the cells with 100 nI fixation buffer (either nucleus or cytoplasm system) for 30 min at room temperature in dark. For nucleus fixation, mix one portion of Nucleus Fix 1 with 3 portions of Nucleus Fix before use.
4. Centrifuge at 1500 rpm and remove the supernatant.
5. Prepare the proper amount (3 folds of the fixation buffer) of the working permeabilization buffer by diluting Permeabilization Buffer stock solution 10 times with distilled water. Wash the samples with 100 pi permeabilization buffer.
6. Incubate with 100 pi permeabilization buffer for about 30min at room temperature, centrifuge and discard the supernatant.
7. Re-suspend with 100 pi permeablization buffer, followed by adding 10 pi 1:40 diluted AF647-anti-SAM antibody included in the kit. Incubate at 4 °C for about 60 min.
8. Wash each sample with 1ml PBS 2-3 times and re-suspend it with 0.5 ml PBS.
9. Run each sample through a Flow Cytometer.

S-adenosylmethionine, a sulfur-containing molecule, is a biologically very active metabolite and its important roles in methylation as a key methyl donor, transsulfuration and aminopropylation have been well understood. SAM is an intrinsically unstable molecule with molecular weight of 398.44, and its optical density maximum of 258-260 nm is not a distinguished absorption, accurate and timely determination of its concentration in various biological fluids and tissues has always been a challenging task. Therefore, finding a way that can quickly and specifically determine the amount of SAM becomes essential. With the advent of specific anti-SAM monoclonal antibodies, measure SAM from living cells directly, quickly and sensitively becomes possible with flow cytometry. Alexa Fluor® 647conjugated mouse anti-SAM antibody has been used (Refer to Cat# MAF00201) after cells are fixed and permeabilized. In this kit, both cytoplasm and nucleus fixation/permeabilization buffers have been include. With nucleus fixation/permeabilization system, all targets from cytoplasm and nucleus will be detected, whereas with cytoplasm fixation/permeabilization buffer, only cytoplasmic portion of the target is detected.

Components not included in the kit: Distilled water; PBS pH 7.4; Flow Cytometer; Centrifuge