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FAM FLISP FSFCK Serine Protease Assay


Detect active serine protease enzymes with the FAM-FLISP Serine Protease Assay. This in vitro assay employs the green fluorescent label carboxyfluorescein (FAM), which is linked to a 6 carbon spacer, phenylalanine, and a chloromethyl ketone (CMK) reactive group, forming the fluorescent inhibitor probe, FAM-Spacer-Phe-CMK. The 6 carbon hexanoic spacer is designed to reduce steric hindrance of the single amino acid phenylalanine target. FAM-FLISP labels active chymotrypsin-like serine proteases in living cells. Analyze samples using fluorescence microscopy, fluorescence plate reader, or flow cytometry.

Catalog # Size Quantity Price
963 25 Tests $185.00
964 100 Tests $510.00
Catalog #: 963, 964 Categories: , ,

Serine proteases are a family of proteolytic enzymes defined by the presence of a serine residue at the active center of the enzyme. Activated serine proteases play major roles in several different functions including: apoptosis, markers of tumor malignancy, diagnostic and prognostic indicators of breast carcinomas and neck and head carcinomas. Serine protease activity is also altered in a variety of other cell-mediated diseases related to transplant rejection and infections. The most characterized enzymes of this type are trypsin and chymotrypsin. All living cells have a base level of chymotrypsin-like enzyme activity which will vary with the physiological state of the cell as well as by cell type. Because of their supporting role in the apoptotic process, serine protease activity will be greater in apoptotic cell populations compared to healthy cells of the same cell type. Activation of caspases is upstream and likely a prerequisite for activation of serine proteases. FLISP can be used in combination with FLICA to discriminate serine protease activity and caspase activity in the same cell.

The FLISP reagent FAM-Spacer-Phe-CMK enters each cell and binds to activated chymotrypsin-like serine proteases. Because the FAM-Spacer-Phe-CMK FLISP reagent becomes covalently coupled to the active enzymes, it is retained within the cells, while any unbound FAM-Spacer-Phe-CMK FLISP reagent diffuses out of the cell and is washed away. The remaining green fluorescent signal is a direct measure of the active chymotrypsin-like serine protease activity present in the cell at the time the reagent was added. Cells that contain the bound FLISP can be analyzed by a fluorescence plate reader, fluorescence microscopy, or flow cytometry. Cells labeled with the FLISP reagent may be read immediately or preserved for up to 16 hours using the fixative included in the kit. Unfixed samples may also be analyzed with Propidium Iodide or Hoechst 33342 (also included in the kit) to detect changes in necrosis or nuclear morphology, respectively.

1. Prepare samples and controls.
2. Dilute 10X Cellular Wash Buffer 1:10 with diH2O.
3. Reconstitute FLISP with 50 µL DMSO.
4. Dilute FLISP 1:5 by adding 200 µL PBS.
5. Add 10 µL FLISP to each sample (~500 µL aliquot of cultured cells).
6. Incubate ~ 1 hour.
7. Remove media and wash cells three times: add 1X Cellular Wash Buffer and spin cells.
8. If desired, label with additional stains, such as Hoechst 33342, Propidium Iodide, or an antibody.
9. If desired, fix cells.
10. Analyze with a fluorescence microscope, fluorescence plate reader, or flow cytometer. FAM-FLISP excites at 492 nm and emits at 520 nm

Intracellular serine protease activity was monitored over time in Jurkat cells exposed to 1 µM staurosporine. Cells were treated with staurosporine for 1, 2, 3, and 4 hours to induce apoptosis and increase serine protease activity (green histograms), or were untreated (gray histograms). Samples were stained with FAM-Phe-CMK (FFCK, Kit #946) for 1 hour at 37°C prior to being washed to remove unbound FLISP and then analyzed using an Attune NxT flow cytometer. The amount of serine protease activity detected correlated to the duration of the exposure period; the longer the cells were exposed to staurosporine, the larger the portion of serine protease positive cells found in the sample. Data courtesy of Dr. Kristi Strandberg (ICT 231:75-79).

FLISP FMCK FAM-Phe-CMK Serine Protease

• Reagent name: FAM-Spacer-Phe-CMK (FSFCK)
• Target: Chymotrypsin-like enzymes
• Excitation/Emission: 492 nm / 520 nm
• Method of Analysis: Flow cytometry, Fluorescence microscope, Fluorescence plate reader
• Types of Samples: Cell culture
• Storage: FLISP at ≤ -20°C, all other kit components at 2-8°C
• Shipping: Ships overnight (domestic), International Priority Shipping

Kit 963: 25 Tests
• FAM-Spacer-Phe-CMK, 1 vial, #6149
• 10X Cellular Wash Buffer, 15 mL, #6164
• Fixative, 6 mL, #636
• Propidium Iodide, 1 mL, #638
• Hoechst 33342, 1 mL, #639
• Kit Manual

Kit 964: 100 Tests
• FAM-Spacer-Phe-CMK, 4 vials, #6149
• 10X Cellular Wash Buffer, 60 mL, #6165
• Fixative, 6 mL, #636
• Propidium Iodide, 1 mL, #638
• Hoechst 33342, 1 mL, #639
• Kit Manual