FAM-Phe-CMK Serine Protease Assay Kit

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Catalogue No: abx299010
Price: US$551.00
(Size: 25 tests)

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FAM-Phe-CMK Serine Protease Assay Kit

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Datasheet SDS
FAM-Phe-CMK Serine Protease Assay Kit is an assay kit for measuring FAM-Phe-CMK Serine Protease activity using FAM (Green Fluorescence). Please contact us at [email protected] for further information.

Target FAM-Phe-CMK Serine Protease
Tested Applications IF/ICC, FCM
Recommended dilutions Optimal dilutions/concentrations should be determined by the end user.
Storage Store at 4 °C. Avoid prolonged exposure to light.
Availability Shipped within 3-7 working days.
Note THIS PRODUCT IS FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC, THERAPEUTIC OR COSMETIC PROCEDURES. NOT FOR HUMAN OR ANIMAL CONSUMPTION.
Directions for use Experimental Set-Up:

Note that this kit is used with living cells, which may require periodic maintenance and cultivation several days in advance. Create cell populations that have been exposed to experimental treatment and cells that have received no treatment and vehicle only. The density of suspension cells during treatment should not exceed 1 × 106 cells /ml as cells may begin to enter apoptosis. The recommended staining is 10 µl of 50X probe per 490 µl of cells at a concentration of 5 × 105 cells /ml, however this may vary depending on the cells and treatment used. Testing different concentrations of Autophagy Probe Red is recommended. Include appropriate controls, such as:
  • Unlabeled treated (experimental conditions) and untreated cells
  • Labeled treated, untreated and vehicle (for instance DMSO) treated cells
  • As a positive control, several treatments can be used. Starvation in Earl's Balanced Salt Solution (EBSS), Hank’s Balanced Salt Solution (HBSS) or exposure to Rapamycin (mTOR inhibition) can induce autophagy. Exposure to Chloroquine or Bafilomycin A will inhibit fusion of autophagosome with lysosomes and lysosomal degradation.
  • A common pool of cells should be used to generate both the positive and negative control populations for Autophagy Probe Red and should contain similar quantities of cells

Recommended Procedure:

  1. Prepare samples (3-5 × 105 cells /ml) and appropriate controls including positive and negative.
  2. Optional wash step: depending upon the conditions used to induce autophagy, it may be necessary to wash the cells (using PBS containing 5% BSA; or cell culture media) and resuspend in fresh culture medium prior to staining.
  3. Reconstitute 1 vial of Autophagy Probe Red with 100 µl of DMSO to prepare a 250X stock solution (bluish-purple color), which can be aliquoted and stored in the dark at -20 °C for up to 6 months. Immediately before use, prepare a 50X solution by adding 400 µl of PBS to 100 µl of 250X Autophagy Probe red solution. The 50X solution should be used within 30 minutes of preparation.
  4. Transfer cells to fresh tubes, then add 50X Autophagy Probe Red at a ratio of 1 ml sample:50 µl of 50X Autophagy Probe Red (e.g. add 10 µl of 50X Autophagy Probe Red to 490 µl of cells). The ratio of Autophagy Probe Red may need to be optimized depending on experimental conditions and cells used.
  5. Suspension Cells: Incubate at 37 °C in the dark. The incubation time can range from 30 min to several hours depending on the experiment. Gently resuspend the cells every 20 min to ensure even distribution of Autophagy Probe Red.
    Adherent Cells: Trypsinize cells and neutralize the trypsin using standard protocols. Add 2 ml of wash buffer (PBS containing 5% BSA; or cell culture media), gently mix, incubate in the dark for 5 min at room temperature or at 37 °C, then centrifuge cells at 300 × g and discard the supernatant. Repeat this wash step twice.
  6. Centrifuge cells at 200 × g and discard the supernatant.
  7. Resuspend in 500 µl of wash buffer (PBS containing 5% BSA; or cell culture media). Repeat until the samples have been washed 3 times.
  8. Resuspend in 500 ml of wash buffer. Samples can be fixed at this point if required. Add the fixative at a ratio of 1:5 to 1:10.
  9. Analyze samples by measuring the fluorescence using a green/yellow laser equipped with the appropriate filter. Autophagy Probe Red excites at 590 nm and emits at 620 nm.
Research Articles on FAM-Phe-CMK Serine Protease


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