Instruction manual for the angiotensin-converting enzyme 2 (ACE2) active fluorescence detection kit

ACE2 Activity Fluorometric Assay Kit is a rapid and highly sensitive fluorescence test kit for the rapid and sensitive detection of the activity of Angiotensin I Converting Enzyme 2 or Angiotensin II Converting Enzyme (ACE2

The pneumonia epidemic caused by the new coronavirus at the end of 2019, from the beginning of 2020, began a global pandemic, and the number of infections rose rapidly, causing global concern

ACE2 is a zinc-containing metallocarboxypeptidase structure consisting of a signal peptide, a transmembrane domain, and a metalloproteinase activity site

ACE2 plays an important and complex role

The Angiotensin-converting enzyme 2 (ACE2) active fluorescence detection kit uses the fluorescence resonance energy transfer (FRET) method, and its detection principle is as follows

MCA is a fluorescent donor (Donor), Dnp is a fluorescent acceptor (Acceptor) or called quencher , the absorption spectrum of these two fluorophores has a certain overlap, when the distance between the two fluorophores is suitable (generally 7-10 nm), the fluorescence energy is transferred from the donor to the acceptor, resulting in the fluorescence intensity of the donor fluorescent molecule itself decays

Figure 1.

This kit is flexible in use, fast in detection and has a wide range of applications

The MCA Standard is provided in this kit and has a good linear relationship

For 96-well plate inspections, this kit can perform 100 tests in small package P0319S and 500 tests in medium package P0319M

Packing List:

-20 °C storage, valid

The Assay Buffer, Substrate, and MCA Standard (10mM) need to be completely thawed and balanced to room temperature before use, otherwise the results

If the sample is prepared using the Lysis Buffer provided in this kit, and the amount of sample added is within the recommended range of this kit, the pH of the reaction system can be ensured to be within the appropriate range
.
If you use a self-formulated lysate, make sure that the pH of the reaction system after adding the sample is between 6.
5-7.
0, or that the pH of the sample is between 6.
5-7.
0, otherwise the signal value and stability
of the test result may be affected.

When using smaller reagents for the first time, it is recommended to centrifuge for a few seconds to allow the liquid to settle at the bottom of the tube before use
.
Frozen reagents must be completely melted and mixed before use
.

Although the Positive Control (10X) in this kit has been tested to freeze-thaw 5 times and has no effect on the activity, in order to achieve good detection results, it is recommended to properly pack and store
it during the first use.
It is normal for Positive Control (10X) to have a small amount of insoluble matter after melting
.
After dissolving as fully as possible, centrifuge and take the supernatant and use it
.

Although the ACE2 substrate in this kit has high specificity, it is still possible that there are some proteases that can cleave the substrate, and further validation
with the ACE2-specific inhibitor MLN-4760 (SF1197) is recommended.

96-well blackboards are recommended for testing, and it is recommended to purchase all-black 96-well cell culture plates (FCP966).

If the cells, tissues and other lysis samples are stored at 4 °C, the storage time for ACE2 activity detection should not exceed 2 days, otherwise the accuracy
of the detection results will be affected.
Usually, cells, tissues and other lysis samples should be stored at -20 °C, and -80 °C is better stored
.

This product is limited to the scientific research of professionals, and should not be used for clinical diagnosis or treatment, should not be used for food or medicine, and should not be stored in ordinary residences
.

For your safety and health, please wear a lab coat and wear disposable gloves for operation
.

Instructions for use:
1.
Preparation
of samples.

a.
Preparation
of blood samples.

For serum samples, the whole blood is placed at room temperature such as 25 ° C for 30 minutes to 2 hours, do not shake vigorously to avoid hemolysis, after the whole blood naturally coagulates and precipitates the serum, centrifuge at about 1000-2000 ×g at 4 °C for 10 minutes, take the yellow supernatant to obtain the serum, pay attention not to aspirate the white or pale yellow precipitate; For plasma samples, the whole blood is anticoagulated with heparin or EDTA, centrifuged at about 1000-2000 ×g at 4 °C for 10 minutes, and the yellow or yellow supernatant is taken to obtain plasma, taking care not to aspirate the pellet
.
Serum and plasma should be placed on ice and, if not immediately detected, can also be packed and stored at -20 °C or -80 °C
for a short time.
For frozen samples, thaw before testing and store in an ice bath for later use, which must be mixed well
before use.

b.
Preparation
of cell or tissue samples.

For cultured adherent cells, PBS (C0221A) is washed once and the residual liquid
is aspirated.
For cultured suspended cells, first properly centrifuge (e.
g.
, 100-500 × g, 5 min) to collect the cells into a centrifuge tube, discard the supernatant and aspirate the residual liquid
.
Add Lysis Buffer at the ratio of 100-200 μl of Lysis Buffer per 1 million cells, blow appropriately, and ice bath for 5-10 min to fully lyse the cells
.
Centrifuge at approximately 12,000 × g for 3-5 min at 4 °C and take the supernatant for subsequent detection
.
For tissue samples, homogenize
at the ratio of adding 100 μl of Lysis Buffer per 10 mg of tissue.
Centrifuge at approximately 12,000 × g for 3-5 min at 4 °C and take the supernatant for subsequent detection
.
All of the above operations need to be operated at 4°C or on ice
.
Prepared cell or tissue samples can be frozen at -20 °C or -80 °C if they cannot be detected
immediately.
2.
Preparation
of the kit.

Reagents such as Assay Buffer, Substrate, and MCA Standard are equilibrated to room temperature and mixed separately
.
Positive Control (10X) is stored in an ice bath and should be stored
immediately after use in accordance with the conditions required by the kit.
3.
Preparation
of positive controls.

Take an appropriate amount of Positive Control (10X) and add Assary Buffer to dilute
The Polymer Control (10X) 10x.
For example, take 5 μl of Positive Control (10X), add 45μl of Assay Buffer, mix well, and get 50μl of Positive Control (1X
).
For 96-well plate inspections, typically 10 μl of Positive Control (1X) per well is sufficient
.
4.
Setting of MCA standard curve.
Take 2 μl MCA Standard (10 mM), add 198 μl of Assay Buffer, mix well, that is, 200 μl of 100 μM MCA solution, take 0, 2.
5, 5, 10, 20, 30, 40, 50 μl of 100 μM MCA solution into the 96-well plate, and make up to 100 μl with Assay Buffer, at this time, The MCA concentrations and amounts of substances in each well of the MCA standard curve were 0, 2.
5, 5, 10, 20, 30, 40, 50 μM or 0, 0.
25, 0.
5, 1, 2, 3, 4, 5 nmol
, respectively.
You can also set the appropriate MCA concentration for the standard curve
.
5.
Setting of the detection system:
Refer to the following table to add the components and samples
of the kit in turn.
For the initial test, the sample to be measured can dilute a series of concentration gradients to ensure that the final detected value is within the linear range of
the standard curve.
The dilution multiple of the sample to be measured is deil
.

Note: For more reliable test results, it is recommended to set up 3 rewells per sample
.
6.
Detection
.

a.
Shake well and mix well for 1-2 min, making sure to mix well
.

b.
Add 2 μl Substrate per well in addition to the MCA standard curve and mix well
.
Note: The reaction will begin immediately after the addition of Substrate, if the number of wells is large, it is recommended to use the row gun operation at low temperatures or use the row gun operation to reduce the error caused by the time difference between the wells to add Substrate, and the mixing operation can be performed
on the culture plate shaker.

c.
Fluorescence determination using a fluoresce microplate reader immediately after mixing
.
Set the fluorescence microplate reader temperature to 37 °C, the excitation wavelength is 325 nm, and the emission wavelength is 393 nm, and the values
are read every 5 or 10 min.

Note 1: The timing of continuous assays can be adjusted according to the enzyme activity of ACE2 in the sample to be measured, but ensure that data above 6 points are obtained
.
For samples with high enzymatic activity of ACE2, the total assay time is recommended to be 20 min or 30 min, and the corresponding assay interval is set to 2 min or 5 min; For samples with very low enzymatic activity of ACE2, the total assay time can be extended by 1 to 2 hours, and the corresponding assay interval is set to 10 or 20 minutes
.

Note 2: If the fluorescent microplate reader does not have a temperature control function, it can also be measured at room temperature, but the enzyme activity is detected at room temperature, at which time the enzyme activity may be lower, and the degree of low temperature will vary depending on
the experimental conditions.
7.
Calculation
.

Calculate the average fluorescence values for each sample well and blank control well, which can be recorded as RFU blank control, RFU positive control, and RFU sample
to be measured, respectively.
RFU，Relative Fluorescence Unit
。 The data of the time point of the MCA fluorescence intensity of the sample group to be measured was selected for analysis, and the time interval of the linear relationship was T, and the change in fluorescence intensity within the time interval T was ΔRFU, that is, ΔRFU = RFU sample to be measured (Time b) - RFU sample to be measured (Time a).
b.
The relative activity of ACE2 of the sample can also be determined by directly comparing the ΔRFU of each sample over a certain period of time, but it is necessary to ensure that the RFU reading does not reach the platform
at the final time point.

c.
The fluorescence value of each well of the standard curve can also be subtracted from the fluorescence value of the zero concentration well of the standard to establish the MCA standard curve
.
By substituting ΔRFU into the standard curve, the amount of MCA generated in the sample during reaction time (recorded as A)
is calculated.
For the standard curve of MCA, refer to Figure 2A, MCA has a good linear relationship
in the range of 0.
02-5nmol (i.
e.
, 0.
2-50μM).
The formula for calculating ACE2 protease activity is as follows:
ACE2 Activity (nmol/min/mg or U/mg) = A× dil/ (Vsample × T × C)
Note: Vsample is the volume of sample to be measured at the time of detection (Vsample = 10 μl = 0.
01 ml);
A is the amount of MCA generated according to the standard curve determined in step 7c (nmol);
dil is the sample dilution multiple in step 5;
C is the sample protein concentration (mg/ml, the detection procedure refers to the BCA Protein Concentration Determination Kit or the Detergent Compatible Bradford Protein Concentration Determination Kit);
T is the reaction time (min)
in step 7a.

Enzyme activity is defined as: at a temperature of 37 °C, the amount of enzyme catalyzed by 1 nmol MCA per minute is defined as one (Unit, U
).

d.
Although the ACE2 substrate in this kit has been relatively specific, it is still possible that some proteases can cleave the substrate, and further verification
using the ACE2-specific inhibitor MLN-4760 (SF1197) is recommended.

Figure 2.
MCA standard curve of the ACE2 active fluorescence detection kit and the detection effect of ACE2-positive control and tissue samples
.
A.
Schematic of
the MCA standard curve for this kit.
B.
Schematic diagram
of the fluorescence intensity of the product by cutting the substrate in different amounts of ACE2 Positive Control (1X).
C.
Detection of ACE2 enzyme activity in different tissues of mice and inhibition of MLN-4760
.
The total protein amount is 10 μg, the inhibitor is MLN-4760 (SF1197), and the final concentration is 500 nM
).
D.
Detection effect of ACE2 Positive Control (1X, 10 μl) and inhibitor MLN-4760 (final concentration of 500 nM) in 293T cell lysate (total protein amount of 22 μg).

The actual test data will vary depending on the experimental conditions, testing instruments, etc.
, and the data in the figure is for reference
only.