Arthus Biosystems -Model FK00301 -Flow Cytometry Assay Kit 1 for S-Adenosylhomocysteine (SAH)
his kit is designed to measure the amount of SAH in the cytoplasm and nucleus compartments of cells conveniently and quickly using low Cytometry (FCM).
Catalog No. FK00301
Packaging size 60 tests
S-adenosylhomocysteine, a sulfur-containing molecule, is an important molecule in methylation and transsulturation. SAH does not have a distinguished absorption at 258-260 nm, 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 SAH becomes essential. With the advent of specific anti-SAH monoclonal antibodies, measure SAH from living cells directly, quickly and sensitively becomes possible with flow cytometry. Alexa Fluor® 488-conjugated mouse anti-SAH antibody has been used (Refer to Cat# MAF00301) 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
- Prepare the cells to be tested in the amount of 5 -10 x 105/sample in Eppendorf tubes.
- Centrifuge the cell suspension at 1500 rpm for 5 min, and discard the supernatant.
- Fix the cells with 100 pi 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.
- Centrifuge at 1500 rpm and remove the supernatant.
- 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.
- Incubate with 100 pi permeabilization buffer for about 30min at room temperature, centrifuge and discard the supernatant.
- Re-suspend with 100 pi permeablization buffer, followed by adding 10 pi 1:5 diluted AF488-anti-SAH antibody included in the kit. Incubate at 4 °C for about 60 min.
- Wash each sample with 1ml PBS 2-3 times and re-suspend it with 0.5 ml PBS.
- Run each sample through a Flow Cytometer.