FAM FLISP FLCK Serine Protease Assay

$185.00$510.00

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 the target amino acid leucine, and a chloromethyl ketone (CMK) reactive group, forming the fluorescent inhibitor probe, FAM-Leu-CMK. 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
949 25 Tests $185.00
950 100 Tests $510.00
Catalog #: 949, 950 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-Leu-CMK enters each cell and binds to activated chymotrypsin-like serine proteases. Because the FAM-Leu-CMK FLISP reagent becomes covalently coupled to the active enzymes, it is retained within the cells, while any unbound FAM-Leu-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 detected in Jurkat cells using ICT’s green FAM-FLISP serine protease inhibitor FLCK (Kit #950) and cells with compromised membrane activity were identified using Propidium Iodide. Cells were untreated (A.), or were exposed to 1 µM staurosporine for 4 hours to induce apoptosis and increase serine protease activity (B.). During the final hour of treatment, samples were stained with FAM-Leu-CMK for 1 hour at 37°C, and then were washed to remove any unbound FLISP. After wash steps, samples were stained with PI for 15 minutes at room temperature, and then were analyzed using an Attune NxT flow cytometer. Staurosporine treatment resulted in an increase in the median fluorescence intensity for both FLISP (BL1-H, x-axis) and PI (YL1-H, y-axis). Data courtesy of Dr. Kristi Strandberg (ICT 231:80-81).

FLISP vs PI Image FLISP

• Reagent name: FAM-Leu-CMK (FLCK)
• 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 949: 25 Tests
• FAM-Leu-CMK, 1 vial, #6148
• 10X Cellular Wash Buffer, 15 mL, #6164
• Fixative, 6 mL, #636
• Propidium Iodide, 1 mL, #638
• Hoechst 33342, 1 mL, #639
• Kit Manual

Kit 950: 100 Tests
• FAM-Leu-CMK, 4 vials, #6148
• 10X Cellular Wash Buffer, 60 mL, #6165
• Fixative, 6 mL, #636
• Propidium Iodide, 1 mL, #638
• Hoechst 33342, 1 mL, #639
• Kit Manual