Fluorogenic Substrate Assay for tPA Activity

Youssef Farhat, Written 1/29/14, Last updated 1/1/15

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Introduction

Fluorogenic substrates are effective tools for assessing the activity of proteases in media, cell or tissue lysates.  Proteases, such as tPA, are scissor-like proteins whose job is to cut certain amino acid sequences that exist within other proteins, such as plasminogen.  Fluorogenic substrates (also known as FRET substrates) are designed with an amino acid sequence that is cleavable by the protease of interest.  They are also designed in such a way that when a protease cuts the substrate, a fluorescent dye gets released and starts emitting a fluorescent signal.  The amount of signal detected is therefore directly related to how much protease activity there is in a given sample.

Here, we will use a fluorogenic substrate that is specifically cleaved by tPA (tissue Plasminogen Activator).

Note: It is good practice to keep fluorescent substrates protected from light as much as possible.  This can be achieved by covering containers with aluminum foil and working with minimal lighting.

Materials

• SensoLyte® AMC tPA Activity Assay Kit (Anaspec, #72160). This kit includes:
  • tPA substrate (Ex/Em of 354/442 nm)
  • 10 mL of 2X Assay Buffer
  • A few other components which are not going to be used in this protocol, including a tPA standard which we will not use.  The actual amount of tPA in the standard provided with this kit is reported in arbitrary units.

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• tPA Purified Protein (diaPharma, #TC41072)
  • Reconstitute at 100 ng/µL, aliquot 50 µL per tube, and store at -80°C tPA
• Black, flat-bottomed 96-well plates (e.g. Dynex, #3010 or Greiner Cellstar, #655086)
• Deionized water
• Typical lab equipment and supplies such as pipettors, micropipettors, pipette tips, Eppendorf tubes, and a plate reader

Procedure

1. Reagent Preparation

1. Thaw samples and tPA standard (if they were frozen) and keep them on ice.
2. Allow all the other reagents to warm to room temperature (takes ~1 hour)
   1. Note: Keep the substrate protected from light.
3. Dilute 10 mL of 2X Assay Buffer with 10 mL of deionized water.

2. Standard Preparation

1. Dilute a tPA purified protein aliquot (containing 50 µL of tPA at 100 ng/µL) to 10 ng/µL by adding 450 µL to the vial.
2. Fill a set of 8 cDNA strip tubes with 200 µL of assay buffer
3. Take 50 µL of diluted tPA standard and serially dilute it into the first 7 tubes (leaving the last well undiluted to serve as the assay blank), such that the concentration ranges from 2 µg/mL to 0.128 ng/mL

3. Perform the Assay

1. Map out and record the arrangement of your samples using a 96-well plate layout such as the one found here:

• http://protocol-place.com/goodies/printouts/

1. For a full 96-well plate, dilute 50 µL of tPA substrate into 6 mL of Assay Buffer and protect it from light (such as by covering with aluminum foil)

• Note: the manufacturer’s protocol calls for diluting the substrate in only 5 mL of assay buffer, but I found that diluting in 6 mL makes it is easier to add substrate to the 96-well plate, and it has produced good results in my hands.

1. Add 50 µL of each sample and standard to a black, 96-well plate according to your plate layout.

• Note: samples containing a very concentration of tPA may need to be diluted first with assay buffer.  None of the samples I have ever measured required dilution (my samples were media collected from ~70,000 tendon fibroblasts cultured in 6-well plates filled 2 mL of media/well)

1. Bring your plate, multichannel pipette, pipette tips, and the diluted substrate to the plate reader.
2. Set the plate reader to shake the plates for 15 seconds and then make fluorescence measurements with an excitation wavelength of 354 ± 9 nm and emission wavelength of 442 ± 9 nm.  The plate reader can be set to read every 5 minutes for 30 min-1 hour for kinetic measurements, or to read only once at any time between 30 min and 1 hour.

• Sensitivity for the plate reader should be set to an appropriate value.  For our lab’s plate reader, I set the sensitivity to 60 and found that it resulted in a good range of detectable activity (up to 10,000 RFU).

1. Add 50 µL of substrate to the samples and immediately measure fluorescence in the plate reader as indicated above.

References

[1] AnaSpec, “SensoLyte® AMC tPA Activity Assay Kit Data Sheet” <http://www.anaspec.com/servePdf.asp?f=72160.pdf&t=datasheet>

Citing this Protocol

Farhat, Y. “Fluorogenic Substrate Assay for tPA Activity.” Protocol Place. Jan. 2014. Accessed on mm/dd/yyyy. <http://protocol-place.com/>