Cellular urokinase (UROKINASE) activity colorimetric quantitative detection kit product specification

Urokinase Activity Colorimetric Quantification Test Kit Product Manual (Chinese version) Main use

Cellular urokinase (UROKINASE) activity colorimetric quantitative detection reagent is a kind of change aimed at the hydrolysis of urokinase by p-ERG-pNA, a substrate of the tripeptide compound, releasing yellow p-nitroaniline to produce a change in absorbance peak. Colorimetric method to determine the authoritative and classical technical method for enzymatic activity in cell lysis extraction samples. The technology was carefully developed by master scientists and successfully demonstrated by experiments. It is suitable for the detection of urokinase activity in various cell lysis extract samples (animal, human, etc.). The product is strictly sterile, ready to use, simple in operation and stable in performance.

technical background

Urokinase (UK; EC 3.4.2.173), also known as urokinase-type Plasminogen Activator (uPA), is a serine protease present in the blood, extracellular matrix and urine. In the liquid. Urokinase consists of 411 amino acids with a molecular weight of 54 kD and has three domains: a serine protease domain, a kringle domain, and a growth factor domain. The main physiological substrate is Plasminogen, the zymogen of plasmin, and the excision site is Cys-Pro-Gly-Arg⊥Val-Val-Gly. -Gly-Cys. Once activated after excision, plasmin is involved in cellular proteolysis, including thrombolysis and extracellular matrix degradation. Urokinase is involved in tissue remodeling, repair, angiogenesis, and tumor spread. Inhibitors of urokinase are serpins, a Plasminogen Activator Inhibitor (PAI). The synthetic-based tripeptide compound substrate p-EGR-pNA (pyro-Glu-Gly-Arg-p- Nithoaniline; pyroglutamic acid glycyl arginyl-p-nitroaniline) is hydrolyzed by urokinase and released The color of p-nitroaniline was measured by spectrophotometer (405 nm wavelength) to determine the activity of urokinase by measuring the change in absorbance peak. The urokinase response system is:

product content

Cleaning solution (Reagent A) ml

Lysate (Reagent B) ml

Buffer (Reagent C) ml

Negative solution (Reagent D) ml

Substrate solution (Reagent E)

1 product manual

Storage method

Preservation solution (Reagent A) in a 4 ° C refrigerator; the rest is stored in a -20 ° C refrigerator; substrate liquid (Reagent E) to avoid light and repeated freezing and thawing; effective to ensure June

User-supplied

15 ml conical centrifuge tube: container for sample preparation

1.5 ml centrifuge tube: container for sample preparation and storage

Cell scraping rod: for cell detachment

Micro-tabletop centrifuge: for sample sedimentation incubator: for reactant incubation

Enzyme plate or cuvette: a container plate reader or spectrophotometer for sample colorimetric determination: for sample colorimetric analysis

Experimental procedure

Before the start of the experiment, the substrate solution (Reagent E) in the kit at -20 °C in the refrigerator was placed in an ice bath to avoid light. Then do the following.

First, sample preparation

1. Prepare the cultured cells (1 to 5 X 106 cells) in a 25cm2 cell culture flask or 60mm cell culture dish.

2. Carefully add xx ml of cleaning solution (Reagent A) to cover the growth surface.

3. Carefully remove the cleaning solution

4. Use a cell scraping rod to gently scrape off the cells (Note: trypsinization can be used)

5. Add xx ml of cleaning solution (Reagent A) and mix the cells.

6. Move into a pre-cooled 15 ml conical centrifuge tube (note: suspension cells start from this step)

7. Centrifuge in a 4°C benchtop centrifuge for 5 minutes at 300g

8. Carefully remove the supernatant

9. Add xx microliters of lysate (Reagent B) and mix thoroughly

10. Transfer to pre-cooled 1.5 ml centrifuge tube

11. Strong vortex oscillation for 15 seconds

12. Incubate in ice trough for 30 minutes

13. Centrifuge in a 4°C mini tabletop centrifuge for 5 minutes at a speed of 16000g (or 13000RPM, eg eppendorf 5415)

14. Carefully remove 500 μl of supernatant into a new pre-cooled 1.5 mL tube

15. Pipette 10 μl for protein quantification (Note: Bradford Protein Concentration Quantitation Kit is recommended -

YIJI30030.1)

16. Immediately put in -70 ° C to save or place in the ice trough to continue the follow-up operation 2, preparation for determination

1. Prepare the sample to be tested prepared above

2. Set the microplate reader (temperature is 37 ° C): wavelength 405nm, juxtaposition zero, activity determination

1. Mark the 96-well microtiter plate: background control and sample

2. Transfer xx μl of buffer (Reagent C) to the appropriate well.

3. Add xx microliters of negative liquid (Reagent D) or the sample to be tested (50 micrograms of protein) prepared above to the corresponding wells (note: the sample must be clear)

4. Add xx μl of substrate solution (Reagent E) separately

5. Gently shake the 96-well microtiter plate (note: avoid air bubbles)

6. Incubate for 60 minutes at 37 ° C (Note: visible yellow in the well)

7. Immediately put into the microplate reader to detect or transfer to a 100 μl cuvette and test in a spectrophotometer

8. Activity calculation:

(1) Microplate reader detection

Unit = micromolar p-nitrophenol / minute

(2) cuvette detection

Unit = micromolar p-nitrophenol / minute

Precautions

1. This product is 21 operations, including background control

2. Wear gloves when handling

3. Only 1 time for background determination during system operation

4. The sample should be clarified and avoid repeated freezing and thawing

5. The measured value changes from low to high; the measurement lasts for 60 minutes.

6. After the colorimetric determination, the cuvette must be thoroughly cleaned.

7. Sample measurement 60 minutes reading is higher than 0 minutes reading indicates enzyme activity

8. It is recommended that the sample protein concentration to be tested be 50 μg/5 μL; if the sample enzyme activity is too low, the reaction incubation time can be extended to 16 hours; secondly, the sample size is increased (the company provides the Bradford Protein Concentration Quantitation Kit YIJI30030.1) )

9. If the concentration of the sample to be tested is too high or too low, adjust the sample concentration.

10. Urokinase unit activity is defined as the ability to excise 1 micromole of tripeptide substrate per minute at 37 ° C, pH 7..5

The amount of enzyme required for p-ERG-pNA as an active unit

11. The company provides a series of cell protease detection reagent products.

Quality Standard

1. This product has been certified to be stable.

2. This product has been identified and sensitive